GSM3227901: INPUT earlyS rep2; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Input control
Antigen
Input control
Cell type
Cell type Class
Uterus
Cell type
HeLa
Primary Tissue
Cervix
Tissue Diagnosis
Adenocarcinoma
Attributes by original data submitter
Sample
source_name
HeLa S3 cells
description
cervical carcinoma
ezh2i treatment
Non treated
cell cycle
G1/S (Immediately after release)
spike-in
NO
labelling
Non labelled
antibody
none
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Fixed cells were lysed during 20 min in ice-cold lysis buffer (100mM NaCl, 66mM Tris HCl pH8, 5mM EDTA, 0.3% SDS, 1.6% Triton X-100) supplemented with proteases inhibitors, passed through a 21G needle and sonicated using a Bioruptor nextGen (Diagenode) to obtain chromatin frgament size of 250-500bp. Sonicated chromatin was centrifuged at 14000rpm at 4ºC for 10 min and the supernatant used for subsequent steps. Chromatin associated to each histone mark was obtained by incubating sonicated chromatin with the corresponding antibody. In parallel, and independently, Drosophila S2 cells were fixed, lysed and sonicated as described for Hela S3 cells. After sonication, HeLa S3 input chromatin required for the analysis of all time-points from the same time course experiment was mixed with Drosophila S2 chromatin (0.025% of total chromatin). Chromatin associated to each histone mark was obtained by incubating sonicated chromatin with the corresponding antibody. Libraries were costructed using the KAPA Hyperprep kit following manufacturer's instruction, except that 1.25 µM of Illumina compatible indexed adapters (Pentabase) were ligated to A-tailed DNA. Following adapter ligation biotin-TEG-azide (Berry&Associates) was linked to EdU in Click reaction. Biotinylated products were captured using MyOne Streptavidin T1 beads (Thermo Fisher Scientific) and subjected to PCR amplification following the KAPA Hyperprep kit protocol.