protocol

hic & rna

Posted by Yajin on February 20, 2021

Protocol for constructing scRNA-seq and scHi-C library

updated by Y.MO on 2021/02/25

试剂

名称 母液 储液 备注
PBS     公用
SUPERRase Inhibitor      
Protease Inhibitor      
Enzymatics RNase Inhibitor     HXY
BSA 15% -20℃ 15%, -20℃ 公用
FA 37%，4℃ 37%, 4℃ 公用
Glycine 2.5M母液 2.5M, 4℃ 公用
RNase-free water     公用
Nuclease-free water   细胞间4℃ 公用
Tris-HCl pH7.5 1M 1M, 4℃ ZZN
Tris pH8.0 1M 1M, 4℃ DZH
DpnⅡ     师兄定新的
NaCl 5M, 4℃ 1M, 4℃ HXY
EDTA 0.5M, 4℃ 0.5M, 4℃ HXY
EgTA 0.5M, 4℃ 0.5M, 4℃ HLJ
NP-40   1%, 4℃ 公用
Triton X-100   10%, 4℃ 公用
SDS 10%, 4℃ 1%, 4℃ WQJ
10X NEBuffer 3.1   Hi-C公用 & 细胞间4℃ DZH
T4 DNA Ligase Buffer   Hi-C公用 公用
T4 DNA Ligase   Hi-C公用 公用
HpyCH4V     DZH
dA-tail reaction buffer     DZH
Klenow Fragment   Hi-C公用 公用
dNTP (RT用)     HXY
RT primer with biotin tag     HXY
PEG 6000     公用
Maxima H Minus Reverse Transcriptase     HXY
MgCl2 1M, 4℃ 1M, 4℃ XK

溶液配置

名称 配置方法
STE buffer 10mM Tris pH 8.0, 50mM NaCl, and 1mM EDTA
PBSI 0.1U/ul Enzymatics RNase Inhibitor, 0.05U/ul SUPERase inhibitor, 0.04% Bovine Serum Albumin in PBS
  10 ml PBSI = 23 ul 15% BSA + 10 ml PBS
Lysis Buffer 10mM Tris-HCl pH7.4, 10mM NaCl, 0.1mM EDTA, 0.2% NP-40 and 1Xproteinase inhibitor, 0.8U/ul Enzymatics RNase Inhibitor
  10 ml Lysis Buffer = 100 ul Tris-HCl (1M, pH7.5) + 100 ul NaCl (1M) + 2 ul EDTA (0.5M) + 2ml NP-40 (1%) + 7.80 ml Nuclease-free water, 每50ul体系加 ul
NIB 10mM Tris buffer pH 7.5, 10mM NaCl, 3mM MgCl2, 0.1% NP-40, freshly added 0.1U/ul Enzymatics RNase Inhibitor, and 0.05U/ul SUPERase RI
  10 ml NIB = 100 ul Tris-HCl (1M, pH7.5) + 100 ul NaCl (1M) + 33 ul MgCl2 (1M) + 2ul EDTA (0.5M) + 1 ml NP-40 (1%) + 7.64 ml Nuclease-free water
Hybridization mix 1X T4 ligation buffer, 0.32 U/ul Enzymatics RNase Inhibitor, 0.05 U/ul SUPERase RI, 0.1% Triton X-100, and 0.25X NIB
2X Reverse Crosslinking Buffer 100mM Tris pH 8.0, 100mM NaCl, and 0.04% SDS
  10 ml = 1 ml 1 M Tris8.0 + 1 ml 1M NaCl + 400 ul 1% SDS + 7.6 ml N-free water
Tween Wash Buffer 5mM Tris-HCl pH 8.0, 1M NaCl, 0.5mM EDTA and 0.05% Tween
   
2X Binding Buffer 10mM Tris-HCl pH 8.0, 2M NaCl and 1mM EDTA
   
1x TE buffer  
   

  1. 300 g 5 min to centifuge sample cells

  2. Add 50 ul PBSI to the cells in & then on ice, 10U ERI, 5U SI

  3. 2.7 ul 37% FA (final conccentration of 1%), RT 5min

  4. Add 5.56ul 2.5M Glycine, 0.7 ul 15% BSA, 5ul 1M Tris-HCl pH 8.0 on ice, RT 5min

  5. 500g 5min, remove supernatant

  6. Wash cell pallets with 1XPBS with 0.5% BSA (67 ul PBS + 33 ul 15% BSA), 500g 5min

  7. Resuspend in 1ml PBSI

Cell Lysis & RE Digestion

  1. 1000g 3 min to remove supernatant

  2. Resuspand in 50ul lysis buffer on ice, on ice 20min

  3. 1000g 3min to remove supernatant

  4. 8ul ddH2O + 2ul 0.1% SDS, 62℃ 10min

  5. 50ul 1X NEBuffer 3.1 wash twice

  6. Add 48ul 1XNEBuffer 3.1 + 2ul (50U) DpnII, 37℃ overnight

Biotin end repair & ligation

  1. 500g 5min to remove supernatant

  2. Wash cells with 1XT4 DNA ligase buffer

  3. Things on the list were added to the nuclei suspension and incubated at 16℃ for 4-18h with rotation.

   
10x T4 DNA ligase reaction buffer 5 ul
100x BSA (10mg/ml) 1.2 ul
400U/ul T4 DNA ligase 1 ul
Water 44 ul

16℃, 4-18h

dA Tailing and Adaptor for barcodes

  1. Add 30 ul of 5000 U/mL HpyCH4V for 4 h at 37°C while shaking at 1200 rpm (文献体系是446 ul含10E7细胞体系)

  2. 900 g 2 min, supernatant was removed

  3. Washed three times with 1X PBS, 1 mM EDTA, 1 mM EgTA, and 0.1% Triton X-100 solution at 900 g for 2 min

  4. Nuclei concentration was assessed by loading 6 µL of the solution into a disposable hemocytometer (4-Chip Disposable Hemocytometer, Bulldog Bio, #DHC-N420, Portsmouth, NH)

  5. Transfer each 5*E5 nuclei into a new 1.5 mL low-bind Eppendorf tube

  6. Add 25 µL dA-tail reaction buffer, 10 µL of Klenow Fragment and fill to 250 µL using nuclease-free H2O, 37°C 1200 rpm 90 min

  7. Add 200 µL 1X PBS, 50 mM EDTA, 50 mM EgTA, and 0.1% Triton X-100

  8. 900 g 2 min and washed twice using 400 µL of 1X PBS, 1 mM EDTA, 1 mM EgTA, and 0.1% Triton X-100 solution

  9. Resuspended in fresh 60 ul 1X PBS, 1 mM EDTA,1 mM EgTA, and 0.1% Triton X-100 solution

Reverse Transcribe

这部分来自share-seq

  1. Add 240 uL of RT mix (final concentration of 1 3 RT buffer, 0.4U/ml Enzymatics RNase Inhibitor, 500 mM dNTP, 10 mM RT primer with an affinity tag, 15% PEG 6000, and 25U/ml Maxima H Minus Reverse Transcriptase)

  2. RT process as below:

     
  50℃ 10Min
3* 8℃ 12s
3* 15℃ 45s
3* 20℃ 45s
3* 30℃ 30s
3* 42℃ 120s
3* 50℃ 180s
  50℃ 5min

  1. Add 300 uL NIB, 1000g 3min to remove supernatant

  2. Wash pellet with 1ml of NIB and 1000g for 3min

  3. Resuspand cells with 4608 ul of hybridization mix (share-seq体系中是 10,000-20,000 cells)

Hybridization and ligation

  1. Add 40 ul Cells in ligation mix to each of the 96 wells contained 10 uL of barcodes

  2. 30min 300rpm at RT with to allow hybridization to occur before adding blocking strands

  3. Add 10 ul of blocking oligo 30min 300rpm at RT

  1. 500g 5min

  2. Add 50 ul NIB to resuspend

  3. Add 50 ul Reverse Crosslinking Buffer, 2ul 20mg/ml protease K and 1 ul SUPERRase RNase Inhibitor; 65℃ overnight

DNA Extraction

  1. 13k rpm for phase block gel

  2. Add reverse crosslinked mix above gel

  3. add equal amount DNA Extraction Buffer (DNA提取液的有机相) to the phase block gel tube

  4. vortex 30s

  5. 13k rpm 5min to collect supernatant

CATALOG
    FEATURED TAGS
    Homework