Sample information curated by ChIP-Atlas

Antigen

Antigen Class
RNA polymerase
Antigen
RNA polymerase II

Cell type

Cell type Class
Blood
Cell type
REH
Primary Tissue
Blood
Tissue Diagnosis
Leukemia Acute Lymphocytic

Attributes by original data submitter

Sample

source_name
REH cell line
cell line
ALL leukemia cell line REH
chip antibody
Pol2 Ser5P (Abcam, catalog# ab5131)
replicate
1

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
40 million cells were crosslinked with 1% formaldehyde for 10-15 mins. The reactions were quenched by adding glycine to a final concentration of 125 mM, and the cells were centrifuged and washed twice with ice-cold PBS. For each ChIP 10 million cells was used. Nuclei was extracted by washing cell pellet twice with 1 ml of MNase buffer (10mM Tris ph 7,4, 10mM NaCl, 5mM MgCl2, 0,5% IGEPAL CA-630 [Sigma], 1x protease inhibitor cocktail [PIC, Roche], 1 mM PMSF [Thermofisher]). Nuclei were spun down (1500 x g, +4 °C, 5 min) and suspended into 90µl of MNase buffer supplemented with 5mM CaCl2 and 0.1% Triton-X. Different amounts of MNase (0.5-20U; #88216, Thermofisher) was added to the nuclei in 10 µl volume and incubated at 37°C for 10 mins. To stop the reaction, 100 µl of 2xLysis buffer was added to the reaction (1% SDS, 40mM EDTA, 100mM Tris-HCl pH 8.1) and samples were sonicated using Bioruptor (Diagenode) for 5 cycles (30 s – 30 s) to break the nuclei. The lysate was cleared by centrifugation and supernatant was diluted 5 x with RIPA buffer (1X PBS, 1% NP-40, 0.5% Sodium deoxycholate, 0.1% SDS, PIC). The diluted lysate was pre-cleared by rotating for 2 h at 4°C with 60 µl 80% CL-4B sepharose slurry (GE Healthcare; Before use, sepharose was washed twice with TE buffer, blocked for 1h min at room temperature with 0.5% BSA and 20 µg/ml glycogen in 1 ml TE buffer, washed twice with TE and brought up to the original volume with TE). The beads were discarded, and 1% of the supernatant were kept as ChIP input. The protein of interest was immunoprecipitated by rotating the supernatant with 3-5 µg antibody overnight at 4°C. Antibodies against Pol2 Ser2P (ab5095) and Pol2 Ser5P (ab5131) were from Abcam. The Ab was captured using 25 µl blocked Protein G Sepharose® 4 Fast Flow (GE Healthcare, sepharose was blocked rorating overnight at 4°C) and rotating the sample for 2 h at 4°C. The beads were pelleted (1 min, 1000× g, 4°C), the supernatant discarded, and the beads used to bind Ser2P/5P Ab were washed five times with 5X LiCl IP wash buffer (100 mM Tris pH 7.5, 500 mM LiCl, 1% NP-40, 1% Sodium deoxycholate) and twice with TE in 0.45 µm filter cartridges (Ultrafree MC, Millipore). Immunoprecipitated chromatin was eluted twice with 100 µl elution buffer (TE, 1% SDS). The NaCl concentration was adjusted to 300 mM with 5 M NaCl and crosslinks were reversed overnight at 65°C. The samples were sequentially incubated at 37°C for 2 h each with 0.33 mg/ml RNase A and 0.5 mg/ml proteinase K. The DNA was isolated using the ChIP DNA Clean & Concentrator (Zymo Research) according to the manufacturer’s instructions. Sequencing libraries were prepared from collected DNA by blunting, A-tailing, adaptor ligation as previously described (Heinz 2010; MolCell) using barcoded adapters (NextFlex, Bioo Scientific). Between the reactions, the DNA was purified using Sera-Mag SpeedBeads (Thermo Scientific). Libraries were PCR-amplified for 15-16 cycles and size selected for 230-350bp fragments by gel extraction.

Sequencing Platform

instrument_model
Illumina HiSeq 2000

hg38

Number of total reads
24974968
Reads aligned (%)
99.1
Duplicates removed (%)
5.5
Number of peaks
661 (qval < 1E-05)

hg19

Number of total reads
24974968
Reads aligned (%)
98.3
Duplicates removed (%)
7.1
Number of peaks
763 (qval < 1E-05)

Base call quality data from DBCLS SRA