Sample information curated by ChIP-Atlas

Antigen

Antigen Class
RNA polymerase
Antigen
RNA polymerase II

Cell type

Cell type Class
Pluripotent stem cell
Cell type
ES cells
NA
NA

Attributes by original data submitter

Sample

source_name
Mouse embryonic stem cells with human HEK293T cell spike-in, tamoxifen treated, S5P-RNAPII ChIP-seq
tissue
embryonic stem cells
genotype
Kdm2a/b-CXXCfl/fl
spike in cells
human HEK293T cells
replicate
4
treatment
tamoxifen
chip antibody
RNAP_S5P

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
For histone H3 and H3K36me2 ChIP, 1x107 mESCs were crosslinked for 10 min in 1% formaldehyde. Reactions were quenched by addition of 125 mM glycine. The released nuclei were washed twice in PBS, then resuspended in lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl pH 8.1 and 1xPIC) and incubated on ice for 30 min. For KDM2A/B ChIP, 5x107 mESCs were resuspended in PBS and mixed with 2x106 HEK293T cells. Cells were crosslinked in 2 mM disuccinimidyl glutarate (Thermo Scientific) for 45 min at 25°C with gentle rotation, then in 1% formaldehyde for 12.5 min (methanol-free, Life Technologies). Reactions were quenched by addition of 125 mM glycine, and crosslinked cells were resuspended in lysis buffer (50mM HEPES-KOH pH 7.9, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP40, 0.25% TritonX-100 and 1x PIC) and rotated for 10 min at 4°C. The released nuclei were washed (10mM Tris-HCl pH 8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA and 1x PIC) for 5 min at 4°C, and the nuclear pellet resuspended in 1 ml sonication buffer (10mM Tris-HCl pH 8.0, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% sodium deoxycholate, 0.5% N-lauroylsarcosine and 1xPIC). For RNAPII ChIP, 5x107 mESCs were resuspended in PBS and mixed with 4x106 HEK293T cells. Cells were crosslinked for 10 min in 1% formaldehyde. Reactions were quenched by addition of 150 mM glycine, and the crosslinked cells resuspended in FA-lysis buffer for 10 min (50mM HEPES pH 7.9, 150mM NaCl, 2mM EDTA, 0.5mM EGTA, 0.5% NP40, 0.1% sodium deoxycholate, 0.1% SDS, 10mM NaF, 1mM AEBSF, 1xPIC(Roche)). For H3, H3K36me2, KDM2A/B or RNAPII ChIP, chromatin was sonicated using a BioRuptor Pico sonicator (Diagenode), shearing genomic DNA to approximately 0.5 kb. Sonicated chromatin was diluted 10-fold in ChIP dilution buffer (1% TritonX-100, 1mM EDTA, 20mM TrisHCl pH 8, 150mM NaCl and 1x PIC) for H3, H3K36me2 or KDM2A/B ChIP, or in FA-lysis buffer for RNAPII ChIP. Chromatin was pre-cleared for 1 hour with either protein A agarose beads (Repligen, for H3, H3K36me2 or RNAPII ChIP) or protein A magnetic Dynabeads (Invitrogen, for KDM2A/B ChIP) blocked with 1 mg/ml BSA and 1 mg/ml yeast tRNA. For each ChIP reaction, 150 ?g chromatin (KDM2A/B), 300 ?g chromatin (RNAPII) or chromatin corresponding to 1x105 cells (H3 or H3K36me2 ChIP) was incubated overnight with the appropriate antibody: anti-H3 (in house, 15 ?l), anti-H3K36me2 (in house, 15 ?l), anti-KDM2A (in house, 2.4 ?l), anti-KDM2B (in house, 2 ?l), anti-Rbp1-NTD (CST D8L4Y, 15 ?l), anti-Rbp1-CTD-Ser5P (CST D9N5I, 12.5 ?l), anti-Rbp1-CTD-Ser2P (CST E1Z3G, 12.5 ?l). Antibody-bound chromatin was isolated using blocked protein A agarose (H3, H3K63me2 or RNAPII ChIP) or magnetic beads (KDM2A/B ChIP) for 2 hours at 4°C. For H3, H3K36me2 or KDM2A/B ChIP, washes were performed with low salt buffer (0.1% SDS, 1% TritonX-100, 2mM EDTA, 20mM Tris-HCl pH 8, 150mM NaCl), high salt buffer (0.1% SDS, 1% TritonX-100, 2mM EDTA, 20mM TrisHCl pH 8, 500mM NaCl), LiCl buffer (250mM LiCl, 1% NP40, 1% sodium deoxycholate, 1mM EDTA, 10mM TrisHCl pH 8) and two washes with TE buffer (10mM Tris-HCl pH 8, 1mM EDTA). For RNAPII ChIP, washes were performed with FA-Lysis buffer, FA-Lysis buffer containing 500mM NaCl, DOC buffer (250mM LiCl, 0.5% NP40, 0.5% sodium deoxycholate, 2mM EDTA, 10mM Tris-HCl pH 8) and two washes with TE buffer. ChIP DNA was eluted in elution buffer (1% SDS, 100mM NaHCO3) and crosslinks reversed overnight at 65°C with 200mM NaCl and 2 ?l RNase A (Sigma). A matched input sample (corresponding to 10% of original ChIP reaction) was treated identically. The following day, samples were treated with 20 ?g/ml Proteinase K (Sigma) for 2 hours at 45°C and purified using the ChIP DNA Clean and Concentrator Kit (Zymo Research). For H2AK119ub1 and H3K27me3 native ChIP-seq, 5 x 107 mouse ESCs were resuspended in 1ml RSB (10mM Tris-HCl pH 8.0, 10mM NaCl, 3mM MgCl2, 5mM N-ethylmaleimide (NEM)), then lysed by addition of 28 ml RSB containing 0.1% NP40. The released nuclei were washed in RSC buffer (RSB supplemented with 0.25M sucrose and 3mM CaCl2), then resuspended in 1 ml RSC buffer containing 10mM NEM and 1xPIC(Roche). Each sample was then incubated with 150 units of MNase (Fermentas) at 37°C for 5 min, then 4 mM EDTA was added to halt MNase digestion. Following centrifugation at 5000 rpm for 5 min at 4°C, the supernatant (S1) was retained. The pellet was resuspended in 300 µl nucleosome release buffer (10mM Tris-HCl pH 7.5, 10mM NaCl, 0.2mM EDTA, 10mM NEM, 1xPIC), incubated at 4°C for 1 h, then passed five times through a 27G needle using a 1 ml syringe. After centrifugation at 5000 rpm for 5 min at 4°C, the second supernatant (S2) was combined with S1. A small aliquot of S1/S2 DNA was purified and visualized on a 1.5% agarose gel to confirm digestion to predominantly mono-nucleosomes. S1/S2 was diluted 10-fold in native ChIP incubation buffer (70mM NaCl, 10mM Tris-HCl pH 7.5, 2mM MgCl2, 2mM EDTA, 0.1% TritonX-100, 10mM NEM, 1xPIC). For each ChIP reaction, 1 ml of diluted nucleosomes was incubated overnight at 4°C with 5 ?l of anti-H2AK119ub1 (CST D27C4) or anti-H3K27me3 (in-house). Antibody-bound nucleosomes were isolated by incubation for 1 hour at 4°C with protein A agarose beads, which were pre-blocked overnight in native ChIP incubation buffer supplemented with 1 mg/ml BSA and 1 mg/ml yeast tRNA. The beads were then washed four times with native wash buffer (20mM Tris-HCl pH 7.5, 2mM EDTA, 125mM NaCl, 0.1% TritonX-100) and once with TE buffer. ChIP DNA was eluted using 100 ul of elution buffer (1% SDS, 0.1 M NaHCO3), then purified using the ChIP DNA Clean and Concentrator Kit (Zymo Research). DNA from a matched input sample (corresponding to 10% of original ChIP reaction) was also purified.

Sequencing Platform

instrument_model
NextSeq 500

mm10

Number of total reads
25031815
Reads aligned (%)
92.4
Duplicates removed (%)
5.1
Number of peaks
12368 (qval < 1E-05)

mm9

Number of total reads
25031815
Reads aligned (%)
91.9
Duplicates removed (%)
5.3
Number of peaks
12353 (qval < 1E-05)

Base call quality data from DBCLS SRA