Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Histone
Antigen
H3K36me2

Cell type

Cell type Class
Unclassified
Cell type
Unclassified
NA
NA

Attributes by original data submitter

Sample

source_name
Mouse embryonic stem cells & S2 cells (spiked-in)
chip antibody
H3K36me2 (Cell Signaling Tech, #2901)
cell line
ESC line V6.5 1A
cell type
C57BL/6 x 129S4/SvJae F1 embryo-derived embryonic stem cells
genotype
wild-type

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
To obtain a soluble chromatin extract, ~2x10^7 cells were resuspended in 1 mL LB1 (50 mM HEPES, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1x Complete protease inhibitor) and incubated rotating at 4°C for 10 min. Samples were centrifuged, resuspended in 1 mL LB2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1x Compete protease inhibitor), and incubated rotating at 4°C for 10 min. Finally, samples were centrifuged, resuspended in 1 mL LB3 (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na deoxycholate, 0.5% N-lauroylsarcosine, 1% Triton X-100, 1x Complete protease inhibitor), and homogenized by passing two times through a 27-gauge needle. Chromatin extracts were sonicated for 8 min (anti-HA ChIP) or 12 min (anti-histone PTM ChIP) using a Covaris E220 focused ultra-sonicator at peak power 140, duty factor 5, and cycles/burst 200. For histone PTM ChIP-Rx, after centrifugation soluble chromatin was spiked-in with soluble chromatin from Drosophila S2 cells that was similarly prepared and equivalent to 5-10% of the mouse/human cell chromatin. The lysates were incubated with 100 μl Pierce anti-HA beads (Themo Scientific, 88836) or with anti-H3K4me1 (Abcam, ab8895), anti-H3K9me3 (Abcam, ab8898), anti-H3K27ac (Active Motif, 39133), anti-H3K27me3 (Cell Signaling Tech, 9733), anti-H3K36me2 (Cell Signaling, 2901) or anti-H3K36me3 (Active Motif, 61101) antibody bound to 75 μl protein A or protein G Dnya1 magnetic beads (Invitrogen) and incubated overnight at 4°C with 5% kept as input DNA. Magnetic beads were washed with low salt buffer (150 mM NaCl; 0.1% SDS; 1% Triton X-100; 1 mM EDTA; 50 mM Tris-HCl), high salt buffer (500 mM NaCl; 0.1% SDS; 1% Triton X-100; 1 mM EDTA; 50 mM Tris-HCl), LiCl buffer (150 mM LiCl; 0.5% Na deoxycholate; 0.1% SDS; 1% Nonidet P-40; 1 mM EDTA; 50 mM Tris-HCl) and TE buffer (1 mM EDTA; 10 mM Tris-HCl). For ChIP-seq, beads were resuspended in elution buffer (1% SDS, 50 mM Tris-HCl pH 8.0, 10mM EDTA, 200 mM NaCl) and incubated for 30 min at 65°C. After centrifugation the eluate was reverse cross-linked overnight at 65°C. The eluate was then treated with RNaseA for 1 hr at 37°C and with Proteinase K (Roche) for 1 hr at 55°C and DNA was recovered using Qiagen PCR purification kit. KAPA HTP Library Preparation Kit

Sequencing Platform

instrument_model
Illumina HiSeq 4000

dm6

Number of total reads
64997012
Reads aligned (%)
1.7
Duplicates removed (%)
25.8
Number of peaks
756 (qval < 1E-05)

dm3

Number of total reads
64997012
Reads aligned (%)
1.8
Duplicates removed (%)
25.7
Number of peaks
1319 (qval < 1E-05)

Base call quality data from DBCLS SRA