Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Histone
Antigen
H3K36me3

Cell type

Cell type Class
Blood
Cell type
CD4+ T cells
NA
NA

Attributes by original data submitter

Sample

source_name
primary CD4+ T cells
cell type
primary CD4+ T cells
antibody
H3K36me3 (ab9050)
date of sequencing
06 2016

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
20x10^6 CD4+ T cells were washed 1 time in PBS prior to crosslinking with 1% Formaldehyde for 10 minutes at room temperature, followed by termination of the reaction with 125mM glycine on ice. Cell pellet was washed 2 times with PBS at 4C and was lysed in 0.5% NP-40 buffer (10mM Tris-Cl pH7.4, 10 mM NaCl, 3 mM MgCl2, 1 mM PMSF and Protease Inhibitors). For histone ChIPs, obtained nuclei were washed once in the same buffer without NP-40. Nuclei were resuspended in 0.5% NP-40 buffer supplemented with 0.15 % SDS and 1.5 mM CaCl2. Nuclei were incubated at 37 C for 10 minutes prior to addition of Micrococcal Nuclease (16 units of the enzyme), and the reaction was stopped after 7 minutes with 3 mM EGTA. DNA was additionally sheared by sonication (Covaris or Bioruptor, Diagenode) to an average size of DNA fragments below 500 bps. Extracts were then diluted up to 0.01% SDS, 1% Triton-X, 20mM Tris pH 8, 150mM NaCl 2 mM EDTA. Extracts were precleared by 1 hr incubation with protein A/G Magna ChIP beads at 4C and diluted with 5x IP buffer to a final concentration of 140 mM NaCl and 1 % NP-40. Lysate corresponding to 3-4 x 106 million of cells was then incubated with 2-4 µg of the indicated antibody overnight at 40 C, followed by a 2.5 hr incubation with Magna ChIP Protein A/G Magnetic Beads (Millipore). Beads were then washed thoroughly with RIPA150, with LiCl – containing buffer and with TE buffer, RNAse treated for 1 hr at 370C, and Proteinase K treated for 2 hours at 560C. Decrosslinking of protein–DNA complexes was performed by an overnight incubation at 650C. Additional 1 hr of Proteinase K digestion was performed at 560C and DNA was then extracted using Agencourt AMPure XP beads (Beckman Coulter) and quantified by real time PCR. The following antibodies were used ChIP: H3K27ac (ab4729), H3K4me3 (ab8580) H3K36me3 (ab9050) IgG Rabbit (ab46540). ChIP-Seq: Approximately 10 ng of corresponding inputs and ChIP-ed DNA from primary CD4 T cells: H3K27Ac, H3K4me3, H3K36me3, H4K20me1 and H3K9me2 Ips was prepared for the sequencing using NEBNext® Ultra™ II DNA Library Prep Kit

Sequencing Platform

instrument_model
Illumina HiSeq 2500

hg38

Number of total reads
3329245
Reads aligned (%)
81.9
Duplicates removed (%)
11.0
Number of peaks
70 (qval < 1E-05)

hg19

Number of total reads
3329245
Reads aligned (%)
80.8
Duplicates removed (%)
12.4
Number of peaks
91 (qval < 1E-05)

Base call quality data from DBCLS SRA