Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
Rad21

Cell type

Cell type Class
Blood
Cell type
CH12
Tissue
Blood
Lineage
cellLine
Description
B-cell lymphoma (GM12878 analog)

Attributes by original data submitter

Sample

source_name
CH12 B cell line
cell line
CH12
cell type
lymphoma cell line
genotype/variation
WT
chip antibody
Abcam, ab992

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Extraction protocol: ChIP-Seq: Cultured cells were fixed with 1% formaldehyde (Sigma) for 10' at 37°C. Fixation was quenched by addition of glycine (Sigma) at a final concentration of 125 mM. Twenty million fixed cells were washed with PBS and resuspended in 1 ml of RIPA buffer (10 mM Tris [pH 7.6], 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, 1× Complete Mini EDTA free proteinase inhibitor (Roche)) or stored at −80°C until further processing. Sonication was performed using Covaris S2 sonicator at duty cycle 20%, intensity 5, cycle/burst 200 for 30 min or Branson sonifier at amplitude 35%, 12 cycles of 20” sonication and 30” of pause. For native chip, chromatin was digested with MNase (Sigma) in digestion buffer (50 mM Tris-HCl, pH7.6, 1 mM CaCl2, 0.2% Triton X-100, butyrate 5 mM) for 5' at 37°C and dialyzed against RIPA buffer for 2hrs at 4°C. Five micrograms of antibody were incubated with 40 μl of Dynabeads Protein A (or G) for 40 min at room temperature. Antibody-bound beads were added to 500 μl of sonicated or MNase-digested chromatin, incubated at 4°C overnight, and washed twice with RIPA buffer, twice with RIPA buffer containing 0.3M NaCl, twice with LiCl buffer (0.25 M LiCl, 0.5% Igepal-630, 0.5% sodium deoxycholate), once with TE (pH 8.0) plus 0.2% Triton X-100, and once with TE (pH 8.0). Crosslinking was reversed by incubating the beads at 65°C for 4 hr in the presence of 0.3% SDS and 1 mg/ml Proteinase K. ChIP DNA was purified by phenol-chloroform extraction followed by ethanol precipitation. MNase-Seq: 4x10^7 resting and activated B cells were digested with 24 different concentrations of MNaseI. After an initial RT-qPCR analysis and curve-fitting, the MNaseI concentrations for the most representative nucleosomal fraction was determined. The lowest three nucleosomal fractions (which corresponded to MNaseI concentrations of 0U, 0U, and 0.0026U) were pooled together and used as input control. Nucleosomal fractions 16-18 (which corresponded to enzyme concentrations of 0.068U, 0.045U, and 0.03U) were also pooled for further processing. Input samples were sonicated to obtain similar fragment sizes as the MNaseI-digested samples. DNA was then concentrated and each sample was spiked with 0.055mg of l DNA (also sonicated to match the sample size as was input DNA). HiC-Seq: We used the same protocol for in situ HiC libraries as Rao et al., (Cell, 2014 Dec; 159:1-16). 4C-seq: The 4C assay was performed as previously described van de Werken et al. (2012) with minor modifications. Ten million of CH12 B cell line were crosslinked in 2% formaldehyde at 37°C for 10 min. The reaction was quenched by the addition of glycine (final concentration of 0.125 M). Cells were then washed with cold PBS and lysed (10 mM Tris-HCl, pH 8.0, 10 mM NaCl, 0.2% NP-40, 1× complete protease inhibitors [Roche]) at 4°C for 1 hr. Nuclei were incubated at 65°C for 30 min, 37°C for 30 min in 500 μl of restriction buffer (New England BioLabs DpnII buffer) containing 0.3% SDS. To sequester SDS, Triton X-100 was then added to a final concentration of 1.8%. DNA digestion was performed with 400 U of DpnII (New England Biolabs) at 37°C overnight. After heat inactivation (65°C for 30 min), the reaction was diluted to a final volume of 7 ml with ligation buffer containing 100 U T4 DNA Ligase (Roche) and incubated at 16°C overnight. Samples were then treated with 500 μg Proteinase K (Ambion) and incubated overnight at 65°C to reverse formaldehyde crosslinking. DNA was then purified by phenol extraction and ethanol precipitation. For circularization, the ligation junctions were digested with Csp6I (Fermentas) at 37°C overnight. After enzyme inactivation and phenol extraction, the DNA was religated in a 7 ml volume (1,000 U T4 DNA Ligase, Roche). Three micrograms of 4C library DNA was amplified with Expand Long template PCR System (Roche). Thermal cycle conditions were DNA denaturing for 2 min at 94°C, followed by 30 cycles of 15 s at 94°C, 1 min at 58°C, 3 min at 68°C, and a final step of 7 min at 68°C. Bait was amplified with inverse PCR primers as follows: 3RR with DpnII: _4C 5′- GCTTATCTGTAAAGAATGGGTC-3′, 3RR_Csp6i 5′-GGCCTTAGAAGGCTCTGTAC-3′. 4C-amplified DNA was microsequenced with the Illumina platform. mRNA-seq: Total RNA from 10^6 of WT or ZF9-11 CH12 cells was isolated by Trizol extraction. mRNA was then isolated. ERCC-spike-in was added on some samples for quantitative comparison between two conditions. ATACseq: One hundred thousand cultured cells were harvested for each point. Cell lysis and Transposition reaction were immediately performed according to Greenleaf's group protocol (Corces et al., Nat Meth 2017; Buenrostro et al., Nat Meth 2013). Transposed DNA was immediately purified and stored at -20C before library preparation according to the above protocol. CRISPRscreen: Cells were harvested at an early reference time-point (second day after infection) and two late time-points (14 and 21 days after infection). DNA was extracted using DNeasy Blood and tissue kit according to the manufacturer's instructions (#69506, Qiagen). Library construction protocol: ChIP-Seq Library was prepared in Ovation SP Ultralow library system (Nugen). 50 cycles of sequencing data were acquired on the HiSeq 2500 or 3000 (Illumina). HiC-seq: DNA was isolated and prepared for paired-end Illumina sequencing following the genomic DNA protocol. 100 cycles of sequencing data were acquired on the HiSeq 2500 (Illumina). 4C-seq: DNA was prepared for paired-end Illumina sequencing. 50 cycles of sequencing data were acquired on the HiSeq 2500 (Illumina). mRNA-seq: Standard RNA-Seq library preparation was performed following Illumina's RNA-Seq protocol v2. 50 cycles of sequencing data were acquired on HiSeq 2000, 2500 or 3000 (Illumina). ATAC-seq: DNA was prepared for single-end Illumina sequencing. 50 cycles of sequencing data were acquired on the HiSeq 2000 (Illumina). CRISPRscreen: DNA was prepared for single-end Illumina sequencing using staggered GeCKO readout primers (Shalem et al. 2013). 80 cycles of sequencing data were acquired on the MiSeq (Illumina).

Sequencing Platform

instrument_model
Illumina HiSeq 3000

mm10

Number of total reads
32983607
Reads aligned (%)
98.5
Duplicates removed (%)
21.5
Number of peaks
44739 (qval < 1E-05)

mm9

Number of total reads
32983607
Reads aligned (%)
98.2
Duplicates removed (%)
21.5
Number of peaks
44767 (qval < 1E-05)

Base call quality data from DBCLS SRA