題目：Ultra-high-throughput single-cell RNA sequencing and perturbation screening with combinatorial fluidic indexing
期刊：Nature Methods
通訊作者：Christoph Bock
奧地利科學(xué)院分子醫(yī)學(xué)研究中心，維也納，奧地利
人工智能研究所，醫(yī)學(xué)統(tǒng)計、信息學(xué)和智能系統(tǒng)中心，維也納醫(yī)科大學(xué)，奧地利維也納
我們開發(fā)實驗和計算方法，以獲得獨特的視角。例如，我們將下一代測序與生化技巧相結(jié)合，幫助我們繪制各種類型的表觀遺傳修飾圖，并開發(fā)生物信息學(xué)算法，從此類高通量數(shù)據(jù)中推斷相關(guān)的癌癥生物學(xué)。他的研究將實驗生物學(xué)（高通量測序、表觀遺傳學(xué)、CRISPR篩查、合成生物學(xué)）與癌癥、免疫學(xué)和精密醫(yī)學(xué)的計算方法（生物信息學(xué)、機(jī)器學(xué)習(xí)、人工智能）相結(jié)合。 他是10x Genomics公司的聯(lián)合創(chuàng)始人，該公司開發(fā)和制造單細(xì)胞基因組學(xué)產(chǎn)品。他也是單細(xì)胞基因組學(xué)計劃的聯(lián)合創(chuàng)始人，這是一個非營利性組織，促進(jìn)單細(xì)胞基因組學(xué)技術(shù)的開發(fā)和使用。

1 背景

微流液滴發(fā)生器（microfluidic droplet generators）是目前最流行的單細(xì)胞測序技術(shù)平臺。它們具有高通量、直接處理和一致的數(shù)據(jù)質(zhì)量，有助于在基礎(chǔ)生物學(xué)和生物醫(yī)學(xué)研究的許多領(lǐng)域廣泛采用單細(xì)胞RNA-seq (scRNA-seq)。在基于液滴的scRNA-seq中，兩個細(xì)胞被裝載到同一個液滴中，它們的轉(zhuǎn)錄組將被標(biāo)記為相同的細(xì)胞條形碼。因此，在數(shù)據(jù)分析過程中，來自這兩個細(xì)胞的轉(zhuǎn)錄本是不可區(qū)分的，從而產(chǎn)生了可能導(dǎo)致結(jié)果偏倚的cell doublet。為了最大限度地減少cell doublets的數(shù)量，單細(xì)胞懸浮液以非常低的濃度加載到微流體裝置中，使得兩個細(xì)胞不太可能進(jìn)入同一液滴。這種解決方法對于許多應(yīng)用來說已經(jīng)足夠了，但它導(dǎo)致了一個主要的概念限制:大多數(shù)液滴是完全功能的(即它們包含條形碼微珠和逆轉(zhuǎn)錄試劑)，但從未接收細(xì)胞，因此不能產(chǎn)生任何單細(xì)胞轉(zhuǎn)錄組。所以，基于液滴的scRNA-seq試劑的使用率非常低，這導(dǎo)致了該方法的高成本，并使大型研究的成本高昂。
Cell hashing：DNA-labeled antibodies；lipid-tagged indices or expressed genetic barcodes。另外，genotype information derived from the transcriptome sequences can be used to identify and remove cell doublets when samples from genetically different individuals are pooled. 然而，由于大多數(shù)轉(zhuǎn)錄本不攜帶任何細(xì)胞特異性index，因此不可能解析doublet細(xì)胞的轉(zhuǎn)錄譜。因此，細(xì)胞哈?；蚣?xì)胞基因型在提高scRNA-seq通量方面的效用是有限的，需要更強(qiáng)大的方法。

2.問題及目的

問題：loading細(xì)胞數(shù)量少，為避免doublet；其他方法，不能很好地區(qū)別轉(zhuǎn)錄本
目的：對所有轉(zhuǎn)錄本進(jìn)行細(xì)胞特異性條形碼標(biāo)記，然后對微流體液滴進(jìn)行大規(guī)模過載。

3.工作流程

scifi-RNA seq design

4.方案

4.1 上樣量

10x Genomics Chromium system：maximum recommended loading concentration (15,300 nuclei per microfluidic channel).只有16.4%的液滴含有一個或多個核(平均每個液滴含有0.2個核)。值得注意的是，即使100倍的過載(每個通道153萬個核)也會產(chǎn)生穩(wěn)定的液滴乳液，并且不會堵塞微流體系統(tǒng)。當(dāng)我們增加加載濃度時，液滴填充率和每滴核數(shù)以可控的方式增加，液滴填充率高達(dá)95.5%，平均每滴9.6個核(每個通道153萬個核)，液滴直徑高度一致。
Chromium ATAC-seq reagents

Droplet overloading for the Chromium scATAC v.1.0 and v.1.1 Next GeM microfluidic chips

4.2 細(xì)胞/細(xì)胞核固定

4.2.1 Sample preparation and permeabilization

A total of 5 million cells were washed with 10 ml of ice-cold 1× PBS (Gibco catalog no. 14190-094) with centrifugation (300g,5 min, 4 °C) and fixed in 5 ml of ice-cold methanol (Fisher Scientific catalog no.M/4000/17) at ?20 °C for 10 min. After two further washes (centrifugation: 300g,5 min, 4 °C) with 5 ml of ice-cold PBS-BSA-SUPERase (1× PBS supplemented with 1% w/v BSA (Sigma catalog no. A8806-5) and 1% v/v SUPERase-In RNase Inhibitor (Thermo Fisher Scientific catalog no. AM2696)), permeabilized cells were resuspended in 200 μl of ice-cold PBS-BSA -SUPERase (1× PBS supplemented with 1% w/v BSA and 1% v/v SUPERase-In RNase Inhibitor), and filtered through a cell strainer (40 μM or 70 μM depending on the cell size). We then used 5 μl of the sample for cell counting in duplicates on a CASY device (Sch?rfe System) and diluted to 5,000 cells μl?1 with ice-cold PBS-BSA-SUPERase. We immediately proceeded with the reverse transcription step of scifi-RNA-seq.

4.2.2 細(xì)胞核準(zhǔn)備

4.2.2.1細(xì)胞提核方案

Preparation of fresh nuclei （方法類似10x genomics）
5million cells in 500 μl of ice-cold Nuclei Preparation Buffer (10mM Tris-HCl pH 7.5, 10mM NaCl, 3mM MgCl2, 1% w/v BSA, 1% v/v SUPERase-In RNase Inhibitor, 0.1% v/v Tween-20, 0.1% v/v IGEPAL CA-630 and 0.01% v/v Digitonin, followed by 5min of incubation on ice. Lysis of the plasma membrane was stopped by adding 5ml of ice-cold Nuclei Wash Buffer (10mM Tris-HCl pH 7.5, 10mM NaCl, 3mM MgCl2, 1% w/v BSA, 1% v/v SUPERase-In RNase Inhibitor and 0.1% v/v Tween-20). Nuclei were collected by centrifugation (500g, 5min, 4 °C), resuspended in 200μl of ice-cold PBS-BSA -SUPERase, 之后同上。
Preparation of nuclei——to fix
5million cells in 500 μl of ice-cold Nuclei Preparation Buffer without Digitonin and without Tween-20 (10mM Tris-HCl pH 7.5, 10mM NaCl, 3mM MgCl2, 1% w/v BSA, 1% v/v SUPERase-In RNase Inhibitor and 0.1% v/v IGEPAL CA-630, followed by 5min of incubation on ice. Lysis of the plasma membrane was stopped by addition of 5ml of Nuclei Wash Buffer without Tween-20 (10mM Tris-HCl pH 7.5, 10mM NaCl, 3mM MgCl2, 1% w/v BSA, 1% v/v SUPERase-In RNase Inhibitor). Nuclei were collected by centrifugation (500g, 5min, 4 °C),

4.2.2.1細(xì)胞核固定

nuclei were fixed in 5ml of ice-cold 1× PBS containing 1–4% formaldehyde (Thermo Fisher Scientific catalog no. 28908) for 15min on ice. Fixed nuclei were collected (500g, 5min, 4 °C), the pellet was resuspended in 1.5ml of ice-cold Nuclei Wash Buffer without Tween-20 and transferred to a 1.5ml tube. After one more wash with 1.5ml of ice-cold Nuclei Wash Buffer without Tween-20 (500g, 5min, 4 °C), fixed nuclei were resuspended in 200 μl of Nuclei Wash Buffer without Tween-20, snap-frozen in liquid nitrogen and stored at ?80 °C. （猜測：修改方案，應(yīng)該是為了適用于細(xì)胞核保存）
對于scifi-RNA-seq，冷凍樣品在37°C水浴中解凍1分鐘后，立即放在冰上。離心(500g, 5min，4°C)，固定核重懸于250 μ l ice-cold Permeabilization Buffer (10mM Tris-HCl, 10mM NaCl, 3mM MgCl2, 1% w/v BSA, 1% v/v SUPERase-In RNase Inhibitor, 0.01% v/v Digitonin , 0.1% v/v Tween-20 for 5min of incubation on ice。250 μl of Nuclei Wash Buffer without Tween-20 was added per sample, and nuclei were collected (500g, 5min, 4 °C). After one more wash with 250 μl of Nuclei Wash Buffer without Tween-20, nuclei were taken up in 100 μl of PBS-BSA-SUPERase. 之后同4.2.1。

4.3 Detailed scifi-RNA-seq protocol description

4.3.1 Reverse transcription

10,000 permeabilized cells or nuclei: 25 μM; 5min at 55 °C
Maxima H Minus Reverse Transcriptase
(with heated lid set to 60 °C): 50 °C for 10min, 3 cycles of [8 °C for 12s, 15 °C for 45 s, 20 °C for 45 s, 30 °C for 30 s, 42 °C for 2min, 50 °C for 3min], 50 °C for 5min, store at 4 °C. Processed cells or nuclei were washed with ice-cold 1× PBS containing 1% BSA.
酶的對比：Maxima H Minus versus Superscript IV。Maxima H Minus會更好一些。

Maxima H Minus versus Superscript IV

4.3.2 Microfluidic thermoligation barcoding on the Chromium scATAC v1.0 platform/ scATAC v1.1 (Next GEM) platform

準(zhǔn)備sample：以scATAC v1.1 (Next GEM) platform為例，The resulting pellet was resuspended in a mix of 41.9 μl of nuclease-free water, 12 μl of 10x Ampligase Reaction Buffer (Lucigen catalog no. A0102K), 2.3 μl of 100Uμl?1 Ampligase , 1.5 μl of Reducing Agent B and 2.3 μl of 100 μM Bridge Oligo。(直接把連接RT引物和ATAC beads 的oligo 以及DNA連接酶加入10x ATAC beads反應(yīng)體系，加第二輪10x cell barcodes)
純化cDNA：第一步Dynabeads MyOne Silane純化類似10x ；第二步改成 1.0× cleanup with SPRIselect beads，eluting in 22 μl of EB Buffer.
PS：兩個平臺Chromium scATAC v1.0 platform/ scATAC v1.1 (Next GEM) platform，數(shù)據(jù)差異不大。

platform

4.4 Template switching

20 ul of sample from the previous step was mixed with 10 μl of 5× Reverse Transcription Buffer, 10 μl of Ficoll PM-400 (20%, Sigma catalog no. F5415-50ML；maybe起到類似PEG8000左右，可以分割分子), 5 μl of 10mM dNTPs, 1.25 μl of Recombinant Ribonuclease Inhibitor, 1.25μl of 100μM Template Switching Oligo and 2.5 μl of Maxima H Minus Reverse Transcriptase. The template switching reaction was incubated for 30min at 25 °C, 90min at 42 °C, storage at 4 °C and cleaned with a 1.0× SPRI cleanup, eluting in 17 μl of EB buffer.

4.5 cDNA enrichment.

15 ul of sample from the step above was mixed with 33μl of nuclease-free water, 50μl of NEBNext High-Fidelity 2× PCR Master Mix (NEB catalog no. M0541S), 0.5μl of 100μM Partial-P5 primer, 0.5μl of 100μM TSO Enrichment Primer, and 1μl of 100×SYBR Green in DMSO (Life Technologies catalog no. S7563). cDNA was amplified in a thermocycler as follows: 98°C for 30s, cycle until fluorescent signal >1,000 relative fluorescence units (RFU; 98°C for 20s, 65°C for 30s, 72°C for 3min), 72°C for 5min in another thermocycler, storage at 4°C. cDNA was cleaned by one 0.8× SPRI cleanup followed by a 0.6× SPRI cleanup, quantified with a Qubit HS assay (Thermo Fisher Scientific catalog no. Q32854), and 1.5ng were checked on a Bioanalyzer High-Sensitivity DNA chip (Agilent catalog no. 5067-4626 and 5067-4627).

cDNA

4.6 Tagmentation

EZ-Tn5 Transposase (Lucigen catalog no. TNP92110)
Tn5 reaction buffer was prepared: 50mM TAPS (Sigma catalog no. T9659-100G), 25mM MgCl2 (Ambion catalog no. AM9530G); the pH was adjusted to 8.5 and the solution was sterile filtered. Tn5 dilution buffer was prepared: 50mM Tris-HCl pH 7.5, 100mM NaCl , 0.1mM EDTA (Invitrogen catalog no. AM9260G), 50% glycerol (Sigma catalog no. G5516-100ML), 0.1% Triton-X100 (Sigma catalog no. X100-100ML), and supplemented with fresh 1mM DTT (Sigma catalog no. 646563-10x.5ML) before use. To achieve maximum library complexity, the entire sample was processed in several tagmentation reactions with 1ng input each. cDNA was diluted to 0.2ngμl?1with nuclease-free water and 5 μl (1ng) per reaction was distributed into a 96-well plate on ice. A mix of 11.25μl nuclease-free water, 5 μl of 5× Tn5 reaction buffer, 2.5 μl of dimethylformamide (Sigma catalog no. D4551-250ML), and 1.25 μl of freshly diluted i7-only transposome (prepared as described below and diluted 1:4.5 in Tn5 dilution buffer) was added. Reactions were incubated for 10min at 55 °C, then cooled for 1min on ice. The enzyme was inactivated by addition of 2.5 μl of 1% SDS (Sigma catalog no. 71736-100ML) for 5min at room temperature. Next, the volume was brought to 50 μl and the fragmented cDNA was purified with a 1.0× SPRI cleanup, eluting in 17μl of EB buffer.

4.7 Library enrichment.

NEBNext High-Fidelity 2× PCRMaster mix
Partial-P5 primer
barcoded P7 primer
Tm= 65°C
Libraries were cleaned with a 0.7× SPRI cleanup with AMPure XP beads. All wells with the same P7 barcode were pooled and subjected to a 0.8× SPRI cleanup, eluting in 0.2 bead volumes of EB buffer.

library

5.應(yīng)用（CRISPR screen - scifi data）

6. 附錄(需關(guān)注的)

6.1 doublet rates

doublet rates

6.2 不同固定條件，數(shù)據(jù)質(zhì)量

Performance metrics for scifi-RNA-seq experiments using a mixture of human Jurkat cells and mouse 3T3 cells, starting from whole cells permeabilized by methanol, freshly isolated nuclei, and nuclei fixed with 1% or 4% formaldehyde (cryopreserved, re-hydrated, and permeabilized)

a

bcd