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#3chan
chaifootsteps · 7 months
Note
That 4chan thread is from July 2022 and talks about HBOMax in winter, with the thread being a new months before Vov's "summer 2023" video, so we can say that 4chan leak is bullcrap at least on the timing.
Viv also did call out the 3chan screenshots and the Reddit one as being untrue, though she could've been using weasel words to try to discount all of it when there were in fact some problems behind the scenes.
Plus the 4chan one indicated Viv wanted it on Amazon Prime and indicated it would drop on HBOMax, and now it's on Amazon Prime, so either 1. the entire 4chan thread is bullcrap, 2. some elements are true and i was intended for HBOMax, then the merger screwed that up s it went back to what Viv wanted.
Either way I don't doubt something messy happened with the production given the radio silence.
Wouldn't surprise me -- Vivzie loves her weasel words, after all. Also, did the same 4chan anon who claimed to work in the industry actually say the "wait until it drops on HBOMax in the winter" thing? The tumblr anon said they did but I'm not seeing it in any of the 4chan screenshots.
4chan anon phrased everything very cautiously, and everything else they've said has lined up.
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moonkssd · 2 months
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[txt:] Yuri speak to me
[txt:] What the hells going on with you??
[txt] This is Roro, answering for Yuri.
[txt] Bzzzt. She's been arrested for murder.
[txt] She barely avoided getting the chop chop.
[txt] We have contacted Uncle Gio <3 for additional aid.
[txt] Rika<3chan worried?
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mugenfinder · 1 year
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all the chans 0chan, once existed for about 6 years and was taken down. 1chan it's all about trains, not offences, not weird, it's just boring but if you want to talk about trains there you go. 2chan the root of all the chans and most internet culture you come here because your really bored nothing else they made graggle simpson tho. 3chan it is true anonymity and it's FUCKING BORING, like really i'm on the site and the top thread is just a guy asking if he's ugly and the top response is "yes"
2chan/Futaba Channel is the only one I have some knowledge of because of Nijikaku. From what I can tell, it's equivocal to an old, still functioning city with another city (4chan) built on top of it. My knowledge of it is equivocal to the shadows on a cave wall. And it's probably better that way.
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3malefuture · 3 years
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THE FUTURE IS 3MALE
THIS IS MY TRUE GENDER IDENTITY. (You don’t need a knob either) I AM 3. (FREE) I AM 3 x the wo/man you are. I only do 3somes. The future is 3male. You are 3 to join.
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therabiddoge · 6 years
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You all talk about #4chan but none of you posers knows anything about the first three chans
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pixielix · 4 years
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୭̥⋆*。 teacher!au chan
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pairing: teacher!chan + gn!reader genre: fluff word count: 0.6k warnings: none
“h-heeeEEeeeYYy juno!” you call out awkwardly across the elementary school’s sport field with a big wave
you never really knew how to interact with kids
“remember me? i’m one of mummy’s friends, i’m here to take you home” you smile
the boy stares up at you blankly, “i don’t wanna go home yet”
yikes, kids are brutal
“hey hey every good athlete takes a nap at home after a big day” the boy’s teacher interrupts
he extends his hand to you, “i’m juno’s teacher, mr bang. his mother called and told me you’d be here to pick him up, that’s very kind of you” 
his smile is bright and almost childlike in contrast to the strong grip of his handshake
he seems a little young to be a teacher but definitely looks the part
dressed neatly in a striped navy cardigan fitted over a white dress shirt, with his flowy dark brown hair parted softly down the middle
“yEp that’s me, the babysitter” you chuckle nervously
you never really knew how to interact with cute guys either
anyway- we should head off now, juno. say goodbye to mr bang” you reach for the boy’s hand but he yanks it away
“no” he huffs
“how about we decide with a race?” mr bang suggests, “whoever wins decides whether you stay or go home”
juno mumbles some audible hmm’s and umm’s
“okay let’s race!” the boy announces
“let’s do it, we’ll go from this end to the other!”
mr bang turns to you with the same warm smile, mouthing a quick “good luck!”
“wait- we’re really doing this? right now?” you stumble after them as they set out onto the grass
you haven’t raced anyone since you were a kid and even then you never won
“not right now - after stretching! right mr bang?”
“right you are, little man” he grins proudly
mr bang leads the two of you in a series of stretches before setting up your finish line at the other end of the field
“on 3!” he calls out with a loud voice
“1, 2...”
“3!” juno yells prematurely, taking off with a head start
“wh- hey!!!!” you groan as the boy sprints off into the distance
you do everything you can to close the distance between you and the kid, but he moves like a tiny rocket
he’s already halfway through a little victory dance by the time you reach the finish line, stumbling to a stop as your legs threaten to collapse under you
mr bang offers you one of his arms to steady yourself and you accept, only to apply a little too much weight and end up leaning against his chest
“s-sorry” you blush, but he laughs it off
“you.........” you turn to juno, still heaving between each word, “you........ cheated......”
“did not!”
“did..... too.....!”
“juno?” mr bang crosses his arms and gives him a stern look
“..... yeah maybe i did”
“a dishonest victory is no victory at all, you know that”
“yeah i know...…” juno heaves a reluctant sigh and tugs at the fabric of your shirt, “fine you win, take me home”
“oh??- well say a proper goodbye to mr bang then”
“bye mr bang” he bows slightly
“bye juno, ill see you tomorrow!” he replies enthusiastically before turning to you with a calm smile, “it was great meeting you”
“yes! thank you for your help, mr-”
“just chan. call me chan” 
“... chan”
“...…………… gross” juno mutters watching the two of you exchange gooey smiles, before dragging you away by pulling your sleeve
“i hope i can see you again!” chan calls out with a big wave
thanks again for picking up juno from school for me today, you’re an angel! Received at 7:14pm
anytime! seriously! i’m free monday, thursday, and friday afternoons but i can take other days off too! i just love kids!!!!!! Sent at 7:15pm
m.list
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missingsunglasses · 4 years
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Bán xe tải cũ Cửu Long 8t 2016 0978729346 xe tại Hà Nội
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+ Danh sách xe mới nhất tại đây: + Danh sách xe 2 cầu: + Danh sách xe từ 1 tấn: + Danh sách xe từ 2 tấn: + Danh sách xe từ 3 tấn: + Danh sách xe từ 4 tấn: + Danh sách xe từ 5 tấn: + Danh sách xe từ 6 tấn: + Danh sách xe từ 7 tấn: + Danh sách xe từ 8 tấn: + Danh sách xe từ 9 tấn: + Danh sách xe thùng, xe ben, theo từng tỉnh tìm tại đây :
HÃY ĐĂNG KÝ THEO DÕI VÀ ỦNG HỘ KÊNH CÁC BÁC NHÉ NHẬN MUA – BÁN – KÝ GỬI XE Ô TÔ CÁC LOẠI + Zalo : 0773.275.777 ——————– XIN CHÂN THÀNH CÁM ƠN ————– Tag: #xeotocu, #xetaicu, #choxetaixeotocu, #banxetaicu,#xeben,#xeotocu,#choxe, #banxe, #banxetai, #xehowo, #xecuulong, #xehoanghuy, #xedongfeng, #xedongben, #xehuyndai, #xethaco, #xeford, #xetoyota, #xehonda, #xeben, #xehoamai, #truonggiang, #hino, #ben3chan, #xetaiben, #xetai, #xetaithung, #xedaukeo, #maycongtrinh, #mayxuc, #mayui, #xenang, #maydao, #komatsu, #hitachi, #doosan, #huyndai, #otocu, #ototai, #3chan, #4chan, #howo, #chenglong, #thaco, #dongfeng, #taithung, #2cau, #1cau Nguồn:https://missingsunglasses.com/ Xem Thêm Bài Viết Khác:https://missingsunglasses.com/meo-vat
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Bán xe tải cũ Cửu Long 8t 2016 0978729346 xe tại Hà Nội
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+ Danh sách xe mới nhất tại đây: + Danh sách xe 2 cầu: + Danh sách xe từ 1 tấn: + Danh sách xe từ 2 tấn: + Danh sách xe từ 3 tấn: + Danh sách xe từ 4 tấn: + Danh sách xe từ 5 tấn: + Danh sách xe từ 6 tấn: + Danh sách xe từ 7 tấn: + Danh sách xe từ 8 tấn: + Danh sách xe từ 9 tấn: + Danh sách xe thùng, xe ben, theo từng tỉnh tìm tại đây :
HÃY ĐĂNG KÝ THEO DÕI VÀ ỦNG HỘ KÊNH CÁC BÁC NHÉ NHẬN MUA – BÁN – KÝ GỬI XE Ô TÔ CÁC LOẠI + Zalo : 0773.275.777 ——————– XIN CHÂN THÀNH CÁM ƠN ————– Tag: #xeotocu, #xetaicu, #choxetaixeotocu, #banxetaicu,#xeben,#xeotocu,#choxe, #banxe, #banxetai, #xehowo, #xecuulong, #xehoanghuy, #xedongfeng, #xedongben, #xehuyndai, #xethaco, #xeford, #xetoyota, #xehonda, #xeben, #xehoamai, #truonggiang, #hino, #ben3chan, #xetaiben, #xetai, #xetaithung, #xedaukeo, #maycongtrinh, #mayxuc, #mayui, #xenang, #maydao, #komatsu, #hitachi, #doosan, #huyndai, #otocu, #ototai, #3chan, #4chan, #howo, #chenglong, #thaco, #dongfeng, #taithung, #2cau, #1cau Nguồn:https://missingsunglasses.com/ Xem Thêm Bài Viết Khác:https://missingsunglasses.com/meo-vat
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paranoid-rhythm · 7 years
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TRANSLATION: JOKER GAME 2ND YEAR D-CLASS, SAKUMA-SENSEI RETURNS!! PART 2
It’s the long awaited school trip!
Warning: 2226 words long. Lots of screaming. And ghosts???? Also, please don’t post somewhere else, just link back to this post. m(_ _)m
Text inside 「these」means that Odagiri is talking via LINE, and italicized text inside asterisks like * these * means that it’s the characters’ thoughts.
More middle school spy boys shenanigans after the cut!
Part 1 - 第1話 「an activity log of a certain day」 Part 3 - 第3話「All out! Evacuation Drill! 」
第2話「The Night Of The School Trip」
Sakuma: Okay, are you all here? So tomorrow, after breakfast you all should meet, well, since there's only 8 of you, it'll be your free time to explore. Meet up is at 8AM sharp at the front of the inn. Make sure you don't over sleep.
Hatano: Staying in a worn out, sloppy inn like this...
Amari: I heard the other classes are staying in a high class hotel near the station.
Miyoshi: Even during the school trip, they treat us like this…
Jitsui: It’s actually refreshing, isn’t it?
Sakuma: Don’t say that it’s worn out! That’s rude to the staff! Just say that it’s like an antique with a certain charm--…
Fukumoto: What about breakfast?
Sakuma: It seems like they’ll start serving at 7AM, but they won’t be serving it in your room like dinner earlier, so just go to the dining area by yourselves.
Tazaki: It’s a worn--…, charming inn and they do serve meals properly huh?
Odagiri: 「I don’t need breakfast, I just want to sleep until the last minute!」
Kaminaga: I don’t need it too…
Tazaki: Are you sure it’s alright that you’re out of bed, Kaminaga?
Miyoshi: Getting over-excited and eating so much during the all-you-can eat buffet earlier, you just reaped what you sowed.
Sakuma: Every single time we go out for a trip, someone always gets sick. Kaminaga, are you sure that you don’t want to take any medicine?
Kaminaga: Medicines are a bit--…
Fukumoto: Kaminaga has a trauma on medicines and syringes, so he’s no good with them.
Sakuma: I-is that so? Anyway, as stated in the sign outside, our group would be the only customers for today. But do not, DO NOT cause any trouble, okay?
Odagiri: 「Trust, zero! 」
Tazaki: Well, if you just look back on what happened earlier, it’s not surprising that our class aren’t trusted.
Amari: It’s because everyone’s so carefree~
Hatano: I don’t want to hear that from someone like you who didn’t even bring his travel itinerary with him!
Sakuma: Do you have any more questions?
Miyoshi: Yes.
Sakuma: Miyoshi?
Miyoshi: We’re sneaking out to explore the town after this, do you want to come with us, Sakuma-sensei?
Sakuma: DON’T YOU DARE SNEAK OUT, GO TO SLEEP!
Odagiri: 「I Googled the area earlier, but there’s nothing to see here.」
Fukumoto: There’s nothing around here, so there’s no point in going out.
Sakuma: It’s as Odagiri says. It’s better to not think of unnecessary things and promptly go to bed instead. Well then, I’ll be going back to my room next door.
Jitsui: Good night, Sakuma-sensei.
Sakuma: Yeah, good night.
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Jitsui: Not even a grade schooler would sleep at 9 in the evening.
Hatano: Whoa, you change faces so fast!
Tazaki: Now that you think about it, with Kaminaga not being able to talk, it sure is quiet.
Fukumoto: Even though he’s the one who was looking forward to the school trip’s night the most. What a shame.
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Kaminaga: The epitome of the school trip is of course, the night time! It’s a bit clichéd but we could play cards or have a pillow fight! HAHAHA! We could even talk about love! HAHAHA! AHAHAHAHA!
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Kaminaga: I feel so sick…
Odagiri: 「Though he was very energetic during the day, who would’ve thought that he would be found in this miserable state…」
Hatano: Don’t add in some murder mystery narration!!
Jitsui: Kaminaga-san’s predisposition to misfortune is truly amazing.
Amari: Predisposition to misfortune?
Jitsui: Doesn’t he always gets caught up in something unfortunate and ends up getting the short end of the stick? Though at times, he gets incredibly good luck, so I guess that balances it out.
Amari: Oh, that’s right! He won the lottery in the shopping district the other day.
Tazaki: Heh, what an incredible small scale of luck.
Kaminaga: Let me go to the toilet… Ahh–… *falls down*
Fukumoto: Let me help you.
Odagiri: 「 I’ll help too! 」
Kaminaga: Aaaahhh….
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Hatano: And so? What do we do?
Miyoshi: What to do huh… it seems like there’s no point in going out and exploring anyway.
Amari: I brought a lot of stuff! Let’s see, I have here card games and board games, around 5 each. Othello, go, shogi, chess and I also have darts, a table tennis set and oh! I also have a bingo set!
Hatano: You forgot to bring your travel itinerary but you brought all that useless games and stuff?!
Tazaki: Are you trying to start a gaming association or something?
Jitsui: So that was why you brought such a big bag.
Miyoshi: Considering Kaminaga’s state, wouldn’t it be fun to go against him?
Amari: Ah! Since this is a rare chance, I want us to have a pillow fight!
Hatano: EVEN THOUGH YOU BROUGHT ALL THAT STUFF?!
Tazaki: I’m actually envious of your carefree lifestyle.
Amari: It’s hard to think of what you want to do on the fly, so I just prepared for all the possibilities~
Jitsui: Amari-san, you’re saying a good thing, but you didn’t even bring the most important thing, which is the travel itinerary.
Amari: There, there, for now, let’s just have a pillow fight!
Miyoshi: I don’t know why you keep pushing for that, is there even any meaning in having a pillow fight, even now that we’re in middle school? That kind of game should only be for grade school--…
Tazaki: Oh my, I’m sorry~ my hand slipped~
Hatano: Uwah! Oi, oi, just now, you hit him squarely on the face…
Jitsui: And not only just the face, he also hit Miyoshi-san’s precious hair… what an admirable declaration of war!
Amari: Huh? Does this mean that the fight has begun?
Miyoshi: Heeeh… if you really want to do it, then I’ll join you in this pillow fight…
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*Kaminaga throwing up*
Fukumoto: We’ll just be nearby, if you need help, just say so.
Odagiri: 「Live on! 」
Fukumoto: Speaking of which, it seems like you didn’t have motion sickness this time, Odagiri.
Odagiri: 「I brought some medicine, so no problem! 」
Fukumoto: Is that so? Then it’s good to hear.
Odagiri: 「I also brought a plastic bag, a bucket, a towel, and disposable gloves. I’m fully prepared for the worst possible scenario! 」
Fukumoto: I’d rather you be prepared in not throwing up instead.
Such an old inn like this, the darkness is overwhelming, it’s ominous.
Odagiri: 「Scary stories are forbidden! 」
Fukumoto: Oh, right, I’m sorry. We should talk about some other things instead.
Odagiri: 「That helps a lot.」
Fukumoto: I heard this story from Kaminaga the other day…
xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx Kaminaga: I was soooo sleepy that night, so when I sent a LINE message to a girl, I accidentally sent it to Jitsui! And the message I sent was full of heart emoticons! And when I woke up the next day and looked at my LINE account, it was nerve-wracking! How do you think he replied?! Jitsui: Sleep peacefully, F-O-R-E-V-E-R~ (♡) Kaminaga: That was the first time I’ve seen a very scary heart mark!
xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx Fukumoto: …is what he said that happened.
Odagiri: 「That certainly is scary in a different way from earlier…」
Fukumoto: You’re right, let’s switch to a more light-hearted story.
Odagiri: 「Speaking of Kaminaga, I too, have a story from the other day…」
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Kaminaga: And then this store--… hey Odagiri! Are you listening?!
Odagiri: 「Yeah.」
Kaminaga: Ehh… you were totally just reading that thread in 3chan! Listen to my story--… huh? Something’s coming over here…
Odagiri: 「Isn’t it a pigeon? 」
Kaminaga: Oh?! Look, look! It landed on my shoulder!
Odagiri: 「It looks like it’s biting something! 」
Kaminaga: It’s a letter… let’s see… hmm?
Tazaki: Dear Kaminaga, Your pants’ zipper is open. Tazaki. Kaminaga: SAY IT IN PERSON!!!
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Odagiri: 「…is what happened.」
Fukumoto: I see, a carrier pigeon huh? It’s well trained.
Odagiri: 「By the way, Kaminaga’s zipper was indeed, all the way open.」
Kaminaga: H-hey you guys… stop talking about me…
Fukumoto: Do you still feel like throwing up?
Kaminaga: I still feel--… urk!!
Odagiri: 「You’re making me remember that time when I suffered from motion sickness.」
*slow, heavy footsteps coming*
Odagiri: 「Ah! 」
Fukumoto: What’s wrong, Odagiri?
Odagiri: 「I think I heard footsteps…」
Fukumoto: Hm? I didn’t hear anything though--…
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Tazaki: Miyoshi, isn’t it about time that you’ve stopped targeting me? Also, if possible, don’t put hard things inside the pillowcase.
Miyoshi: Huh? You mean I can’t put this remote control inside the pillow~?
Hatano: Of course you can’t!
Amari: I’m already tired of this~ why don’t we play cards?
Hatano: Then, one last hit!
Amari: Oops…
Jitsui: Ouch.
Hatano: Oh shit--…
Jitsui: Oh, Hatano-san, you should be careful of your strength or else it’ll be troublesome, I thought my arm was going to break.
Hatano: I--… I’m truly very sorry!
Amari: Did something happen between you and Jitsui?
Hatano: It’s not really something… anyway if you accidentally do anything to that guy, it’s better to apologize at once. Or else he might curse you with black magic…
Miyoshi: Hm? Get back to your beds!
*sound of running footsteps*
Sakuma: You guys are being noisy! Didn’t I… tell you… to go… to sleep!? They’re asleep? That’s weird… I’m sure I heard sounds coming from here. Was I just hearing things? Or was it… a ghost?! No! That’s definitely not the case! I’m just tired!
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Odagiri: Ah… ah… ah… ahhhh… ah…
Fukumoto: O-Odagiri! Calm down! You’re choking me!
Odagiri: Ah… ah… f-f-f-f-foot…. Footsteps! I--… i-i-it’s coming here!
Fukumoto: I-… I-I’m sure that’s just Sakuma-sensei! T-that’s why let go of my--… Ah! O-Odagiri! Wait!
Odagiri: *panting*
Fukumoto: O-… Odagiri!
Tazaki: What’s with you two? Why are you both out of breath?
Odagiri: Gho… there was….
Amari: Eh? You saw a UFO?!
Odagiri: THERE WAS A GHOST!
Amari: That’s it~?!
Hatano: It’s not just “that’s it~!”!! That’s a serious matter!
Tazaki: Hatano, are you perchance, scared of ghost stories too?
Hatano: I’m not scared! I-I just don’t want to hear about them!
Miyoshi: That actually counts as “scared”, you know.
Jitsui: Fukumoto-san, did you see it as well?
Fukumoto: I didn’t see if it truly was a ghost, but there really was a slow, heavy footsteps from the pitch black corridor that was coming closer…
Hatano: Ack--… GAAAAAAAAAAAAAAAH!!!
Tazaki: Declaring that it’s a ghost just by hearing footsteps, I think that’s just your imagination.
Miyoshi: I disagree.
Hatano / Odagiri: Geh?!
Miyoshi: It was quite a long time ago, but this place used to be a cemetery. It seems like it was torn down to make way for this inn to be built. So I do think that there’s a possibility…
Hatano: No, no, no!
Odagiri: There’s no way--…!!
Miyoshi: There might be a ghost here who lost its place, with its grave torn down and it can’t get free…
Hatano / Odagiri: Ah… ah… aaah…..
Miyoshi: ...looking…. for the person responsible…
Hatano / Odagiri: GYAAAAAAAAAAAAAAAAAAHHHH!!!!!!!!!!
Sakuma: YOU GUUUUUUUUUYS!!!!!!!!!!
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Hatano / Odagiri: GYAAAAAAAAAAAAAAAAAAHHHH!!!!!!!!!!
Kaminaga: Just now… did someone a scream…? Fukumoto? Odagiri? What? What’s happening?
H-hey… F-Fukumoto…. O-…Odagiri…. Where are you…?
???? : Hurry up and move from there.
Kaminaga: AAAAAAAAAAAAAAAAAAARRRRGHHHH!!!!
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Sakuma: Didn’t I tell you guys to not make any noise and to sleep already?! Didn’t I warn you not to make any trouble during the school trip?! If anything reaches Vice-Principal Mutou’s ears, you’ll never hear the end of him threatening to expel you guys again! Seriously! I thought that it was suspicious, that’s why I went out to patrol!
Fukumoto: So those footsteps from earlier was really just Sakuma-sensei, huh.
Odagiri: *sigh of relief*
Sakuma: Huh? By the way, where’s Kaminaga?
Kaminaga: AAAAAAAAAAAAAAAAAAARRRRGHHHH!!!!
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Sakuma: Kaminaga! Get a hold of yourself, Kaminaga!
Kaminaga: Ah…. ah…
Sakuma: It looks like he fainted. For the meantime, let’s carry him back to his bed.
Amari: I’ll help you out!
Miyoshi: I wonder what he saw, to faint in front of the washroom like this.
Fukumoto: Maybe just like us, he mistook Sakuma-sensei’s footsteps as a ghost?
Jitsui: Then that wouldn’t match the timing of when he screamed.
Sakuma: Huh? Wait one moment, while I did patrol near you guys’ room, I did not go near the washroom at all.
Fukumoto: Eh?
Odagiri: Ah--… *faints*
Tazaki: This time it’s Odagiri, huh?
Amari: Hatano, are you alright?
Hatano: Huuh?! What are you talking about?! Why would I be not okay!?
Jitsui: But ever since earlier, you’ve been sticking close to me…
Hatano: S-shut up! H-hey Odagiri! Don’t faint just like that!
Odagiri: ugh….
Hatano: Oi! This guy is hopeless! Oi!
Miyoshi: Sakuma-sensei, there’s this one thing I want to ask. What does the inn’s welcome board say?
Sakuma: “Yuuki Private Academy - Middle School Department and Party”, why, is anything the matter?
Miyoshi: Is that so?
Sakuma: Hmm? Is there a connection between that and Kaminaga fainting?
Miyoshi: Who knows? Is it really the work of a ghost, or maybe it was something much more frightening than a ghost…
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Yuuki: Fainting after seeing a person face…
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missmiumiu · 5 years
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Anal Chem. 2018 Jan 16;90(2):1273-1279. doi: 10.1021/acs.analchem.7b04050. Epub 2018 Jan 3.
Single-Cell RT-PCR in Microfluidic Droplets with Integrated Chemical Lysis.
Kim SC1, Clark IC1, Shahi P1, Abate AR1,2,3.
Author information
1Department of Bioengineering and Therapeutic Sciences, University of California-San Francisco , San Francisco, California, United States.
2California Institute for Quantitative Biosciences (QB3), University of California-Berkeley , Berkeley, California, United States.
3Chan Zuckerberg Biohub , 499 Illinois Street, San Francisco, California, United States.
Abstract
Droplet microfluidics can identify and sort cells using digital reverse transcription polymerase chain reaction (RT-PCR) signals from individual cells. However, current methods require multiple microfabricated devices for enzymatic cell lysis and PCR reagent addition, making the process complex and prone to failure. Here, we describe a new approach that integrates all components into a single device. The method enables controlled exposure of isolated single cells to a high pH buffer, which lyses cells and inactivates reaction inhibitors but can be instantly neutralized with RT-PCR buffer. Using our chemical lysis approach, we distinguish individual cells' gene expression with data quality equivalent to more complex two-step workflows. Our system accepts cells and produces droplets ready for amplification, making single-cell droplet RT-PCR faster and more reliable.
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Cells & chemicals
Jurkat and MCF7 cells are cultured in RPMI-1640 medium supplemented with 5% fetal bovine serum (FBS) and antibiotics at 37°C in the presence of 5% CO2. 0.8–1 million Jurkat/MCF7 cells are collected and washed with cold PBS supplemented with 2% FBS (PBS-F). Cells are centrifuged at 300 RCF for 3 minutes, resuspended in 100 μL of 10 μM calcein violet (Thermo Fisher, C34858) or 25 μM calcein red (C34851) in PBS-F, and incubated on ice for 30 minutes. After cell staining, cells are pelleted, resuspended in 100 μL of PBS-F, mixed together and supplemented with 40 μL (0.2× volume) of Optiprep (Sigma, D1556) for density matching. The alkaline lysis buffer (ALB) contains 200 mM NaOH, 60%(v/v) PEG-200 and 2%(v/v) Triton X-100. It is noteworthy that the addition of PEG-200 increases pH significantly,25thus enabling the use of a lower NaOH concentration during the lysis step. This is important so that the buffer capacity of RT-PCR reagents is sufficient to effectively neutralize the alkaline after the merge step. Water-in-oil droplets are generated in a fluorinated oil (3M, HFE Novec 7500) supplemented with 2%(w/w) 008-FluoroSurfactant (RAN Biotechnologies). The oil phase of collected droplets is swapped with the FC-40 oil (Sigma, F9755) supplemented with 5%(w/w) 008-FluoroSurfactant immediately before thermocycling droplets for RT-PCR. The RT-PCR mix is composed of 1× Reaction Mix and 1× Enzyme Mix (Thermo Fisher, 12574030, SuperScript III One-Step) supplemented with 2.5%(v/v) Tween 20 and 2.5%(v/v) PEG 6,000. The FAM-labeled TaqMan probe specific to CD45 protein (IDT PrimeTime, Assay ID: Hs.PT.53a.2558434) is added to the RT-PCR mix.
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在走廊上罰站打手心
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 我們卻注意窗邊的蜻蜓
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  我去到哪裡你都跟很緊 很多的夢在等待著進行 
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一起長大的約定 那樣清晰 打過勾的我相信
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說好要一起旅行 是你如今唯一堅持的任性
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the-au-queen · 3 months
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rules: in a new post, post the names of all the files in you wip folder regardless of how non descriptive or ridiculous. let people send you an ask with the title that most intrigues them and then post a little snippet or tell us about it. and then tag as many people as you have wips
@hergan416 tagged my YGO account but since my WIPs are all over the place, Imma use this account instead hah Be prepared. I have a lot of WIPs
Additionally; due to the reasons listed above, I will add fandom/ships behind the file names. Italics: drabbles, no plan on publishing, mostly written for friends. Bold: multi-chapters in need of a new chapter.
This is going to be extremely long, so the Read More is more of a warning than anything.
Okay so. To not subject the poor souls I'm tagging to the horror of scrolling through the read more, I will tag them now.
@thenarcolepticone, @ami-v-dragnire, @forloveoflibertea, @moophinz, @egyptiansapphiredragons and @yuugisbarber
DMC exchange but make it a group project (Devil May Cry, Vergil/Dante)
A not entirely useful lesson in history (Yu-Gi-Oh Zexal, Gen)
Tell me how he touched you (One Piece, Robin/Dragon)
Nightly Visit (Yuukoku no Moriarty, Fred/Bonde)
Blind Mermaid AU That's Lacking A Title/Sonar (Hetalia, USUK)
How Angels Are Made (Hetalia, USUK)
The World Is Rotten (But I Need Your Help) (Yu-Gi-Oh VRAINS, Entrust)
some mpreg angst (Yu-Gi-Oh DM, Pride)
A Night With The Spirit (Avatar The Last Airbender, Zukaang)
Don't You Puyo With Me (Hetalia, USUK)
Prompt 1 (Yu-Gi-Oh DM, Gen)
Prompt 2 Yu-Gi-Oh DM, Pride)
(Not) Less Of A Person (Yu-Gi-Oh DM, Pride)
Touch Me (Yu-Gi-Oh DM, Pride)
You, Lovely You (Yu-Gi-Oh VRAINS, Entrust)
Let Me Blur Your Lines (Yu-Gi-Oh VRAINS, Entrust)
Seahorse (Yu-Gi-Oh DM, Pride)
Don't Call Me Daddy (Hetalia, USUK)
Press Me Up Against Your Lips
Into The Kaibaverse (Yu-Gi-Oh DM, Pride)
Fatal Meeting (Yu-Gi-Oh DM, Pride)
Our Transfer Student Can't Be This Cute (Miraculous Ladybug, Adrienette)
Like and Subscribe To My Heart (Hetalia, USUK)
Remnant (Mob Psycho 100, Gen)
Voice Pining or alternatively It's Been A While (Yu-Gi-Oh VRAINS, Datastorm)
I want to see you smile (Yu-Gi-Oh VRAINS, Datastorm)
Crimson Flavoured (Avatar The Last Airbender, Zukaang)
VRAINS Shipweek (Yu-Gi-Oh VRAINS, various)
Do You See Me (Yu-Gi-Oh DM, Pride)
The Spirit Of The Ring (Yu-Gi-Oh DM, Mage)
Play My Little Game (Yu-Gi-Oh VRAINS, Datastorm)
Fake Datastorm December (Yu-Gi-Oh VRAINS, Datastorm)
Roxanne (Hetalia, CanNyo!Eng)
Mirage (Hetalia, USUK)
Time travelling cardverse (Hetalia, USUK)
Seto following the Kaibaverse (Yu-Gi-Oh DM, Pride)
The Legend Never Dies (Hetalia, USUK)
Devoted Christian Halfblood (Percy Jackson, Gen)
Full Moon (Yu-Gi-Oh DM, Pride)
3chan (Yu-Gi-Oh DM, Pride)
People really liked the proposal AU (Hetalia, USUK)
Year of the OTP 2023 (various)
Rosa ft Arthur (Hetalia, Gen)
Belle Smut (Beauty and the Beast, Belle/Beast)
Basically Mona Rules Them All (Hetalia, various)
Unnamed Document (Yu-Gi-Oh DM, Pride)
KaruShuu Drabbles (Assassination Classroom, Karma/Gakushuu)
White Day fic Diurnal (Hetalia, USUK)
Shatter Me (Assassination Classroom, Karma/Gakushuu)
Drabble to adopt (Hetalia, USUK)
YGOME fem!Judai (Yu-Gi-Oh GX, Rival)
The Memory Of A Beloved (Yu-Gi-Oh DM, MokuSera)
Perfect Stranger (Hetalia, USUK)
Out Of The Dark (Yu-Gi-Oh VRAINS, Datastorm)
Money for Love/National Anthem (Hetalia, EngMona/USUK)
Oscar (Hetalia, USUK)
Among Mortals (Hetalia, USUK)
False Prince (AKA lame title) (Hetalia, USUK)
Ice Villain Fire Hero (Hetalia, USUK)
Deaf Werewolf Blind Vampire (Hetalia, USUK)
First Day Out (Hetalia, USUK)
Arthur Kirkland's Special Circumstances (Hetalia, USUK)
BuzzFeed Unsolved (Hetalia, USUK)
About Androids and Ancients (Hetalia, USUK)
Sam? Ham? Sham? (Hetalia, USUK)
Small....Big Blunder (Hetalia, USUK)
Arthur (Hetalia, USUK)
Blind, Deaf, Mute. (Hetalia, USUK)
Cookies and Slumberparties (Hetalia, USUK)
Youtube AU (Hetalia, USUK)
Ancient meets Sci-Fi (Hetalia, USUK)
Small dirty lizard thing (Hetalia, USUK)
Mutualism
More (inacurate) intersex England (Hetalia, USUK)
Self indulgent headcanon thing (Hetalia, USUK)
Sand Planet (Hetalia, USUK)
For Maiden (Yu-Gi-Oh VRAINS, Datastorm)
Comparsion and other hijinks (Yu-Gi-Oh, various)
German prideshipping (Yu-Gi-Oh DM, Pride)
Kissing Babies (all) (Yu-Gi-Oh, various)
Short short drabbles (Yu-Gi-Oh, various)
Drabble Games (Hetalia, Gen, various)
NSFW Drabble Games (Hetalia, various)
Tentacles 'n Stuff (Hetalia, USUK)
Rosa fucks monsters (Hetalia, Nyo!England/monster)
NSFW Request (Hetalia, USUK)
Two (2) of the WIPs listed don't have anything to identify them, as I don't remember what I made them for, and since they're empty docs, I'll just have to count on me remembering at some point in life. *shrugs* Not the first time this happened. I always remember at some point.
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mugenfinder · 1 year
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to answer you no the meme doesn't make any sense 1chan isn't even about communism it's about trains and railroads and thats it, it's just kind of boring but not as boring as 3chan
Okay, I didn't know there is a 1Chan, maybe no one should have Chans at this point.
Maybe we should have 0Chans
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doctorsex69 · 4 years
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ifunny tiktok vine reddit 3chan
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nailahs-freebies · 10 years
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Lastest Tax Free Gifts To Children 3chan auctions
Most popular tax free gifts to children 3chan eBay auctions:
[wprebay kw="tax+free+gifts+to+children+3chan" num="0" ebcat="-1"]
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