Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
Rad21

Cell type

Cell type Class
Blood
Cell type
B cells
NA
NA

Attributes by original data submitter

Sample

source_name
aB30h_wt_Rad21_HU_treated
genotype/variation
WT
tissue
spleen
cell type
activated B cell
chip antibody
abcam; ab992
activation time
activated, 30h, HU

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_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: 4x107 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 at Rao et al., (Cell, 2014 Dec; 159:1-16) Gro-seq: Nuclei was extracted from 8-12 million cells grown on 10 cm plates and after run-on reaction the RNA was extracted with Trizol LS Reagent (Invitrogen, Carlsbad, CA, USA). RNA was treated with TURBO DNase (Ambion), fragmented using RNA Fragmentation Reagents (Ambion) and purified by running through P-30 column (Bio-Rad, Hercules, CA, USA). Fragmented RNA was dephosphorylated with PNK (New England Biolabs, Ipswich, MA, USA) followed by heatinactivation. Dephosphorylation reactions were purified using anti-BrdU beads (SantaCruz Biotech, Santa Cruz, CA, USA) and precipitated overnight. Poly(A)-tailing and cDNA synthesis was performed the next day as described in (Wang et al., 2011). However, for reverse transcription oligos with custom barcodes (underlined) were used : 5'-Phos CA/TG/AC/GT GATCGTCGGACTGTAGAACTCT /idSp/CAAGCAGAAGACGGCATACGA TTTTTTTTTTTTTTTTTTTTVN-3'. After cDNA synthesis, Exonuclease I (New England Biolabs; 30 min) was used to catalyze the removal of excess oligo. Enzyme was inactivated and RNA hydrolyzed by alkaline treatment (100 mM NaOH) and heat (25 min, 95°C). The cDNA fragments of were purified on a denaturing Novex 10% polyacrylamide TBE-urea gel (Invitrogen). The recovered cDNA was circularized, linearized, amplified for 10-14 cycles. The final product was ran on Novex 10%TBE gel, gel purified and cleaned-up using ChIP DNA clean & Concentrator Kit (Zymo Research Corporation, Irvine, CA, USA). 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 106 of WT or ZF9-11 CH12 cells was isolated by Trizol extraction. mRNA was then isolated . local Hi-C: 500ng of Hi-C library (8 cycles of amplification) was mixed with 2.5 µg of mouse Cot-1 DNA and 10µg of salomon sperm DNA in a total volume of 25µl. The sample was heated to 95℃ for 5 minutes. Next the temperature was diminished to 65℃ and the samples incubated for at least 5 minutes at 65℃. In the meantime 6µl of a RNA probe mixture (500ng of RNA probes, 20U of supreRNAseIn) and 33µl of hybridization buffer (10xSSPE, 10xDenhardt buffer, 10mM EDTA, 0.2% SDS) were incubated at 65℃. Next, the hybridization buffer and the RNA probe mixture were added to the sample. The final mix was incubated at 65℃ for 24 hours. Following the hybridization, 50 µl of MyOne Streptavidin T1 beads were washed 3 times with Bind-and-Wash buffer (1M NaCl, 10 mM Tris-HCl, pH=7.5, 1mM EDTA) and then resuspended in 134 µl of Bind-and-Wash buffer and added to the sample. The mixture was incubated for 30 minutes at room temperature with occasional swirling. Next, beads were separated using a magnet and the supernatant discarded. Beads were then washed once with 200µl of low-stringency buffer (1xSSC, 0.1%SDS) and incubated for 15 minutes at RT. Beads were separated and washed 3 times with high stringency. ChromRNA-seq: chromatin RNA fraction was prepared from 3*10^6 cells following the method previously described (Pandya-Jones & Black, RNA, 2009) with some modifications. The pellet was lysed in a cytoplasm lysis buffer (20mM Hepes-KOH pH 7.6, 2mM MgCl2, 10% glycerol, 0.1% NP40, 0.5mM DTT with protease inhibitor, phosphatase inhibitor and RNase inhibitor). The lysate was then layered on top of a sucrose buffer in order to isolate the nuclei fraction from the cytoplasmic one (10mM Hepes-KOH pH7.6, 10mM NaCl, 1.5mM MgCl2, 10% glycerol, 0.5mM EDTA, 0.5mM DTT, 34% sucrose w/v with protease inhibitor, phosphatase inhibitor and RNase inhibitor). ChromRNA-seq: The nuclei fraction was resuspended and lysed in a nuclear lysis buffer (10mM Hepes-KOH pH 7.6, 100mM NaCl, 0.5mM EDTA, 50% glycerol, 0.5mM DTT with protease inhibitor, phosphatase inhibitor and RNase inhibitor). Chromatin was precipitated by adding a Urea c

Sequencing Platform

instrument_model
Illumina HiSeq 3000

mm10

Number of total reads
45451876
Reads aligned (%)
98.8
Duplicates removed (%)
21.1
Number of peaks
64906 (qval < 1E-05)

mm9

Number of total reads
45451876
Reads aligned (%)
98.5
Duplicates removed (%)
21.1
Number of peaks
64985 (qval < 1E-05)

Base call quality data from DBCLS SRA