GSM2988418: Input biotin rep1; 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
cell line
HeLa S3
cell type
cervical carcinoma
ezh2i treatment
Non treated
spike-in
NO
labelling
biotin-dUTP
cell cycle
Mid S (3 h and 15 min after release)
antibody
N/A
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.After ChIP, chromatin was separated from Protein A agarose beads by an incubation with Elution buffer (10 mM Tris HCL, 1 mM EDTA, 2 % SDS, 15 mM DTT, pH 8 supplemented with protease inhibitors) for 30 min at 37 ºC in rotation. Supernatant was diluted 20 times with RIPA and chromatin was then incubated with MyOne Streptavidin T1 beads. For single streptavidin pull-downs, 30 μg of input chromatin was used in the following steps. Briefly, MyOne streptavidin T1 beads were blocked by incubating with 1μg/ml free biotin (Sigma) for 10 min at room temperature. Beads were then washed three times with RIPA buffer and chromatin was pre-cleared with blocked beads for 1 h at 4 ºC. After pre-clearing, supernatant was incubated with new non-blocked T1 streptavidin beads over night at 4 ºC. Beads were then washed for 5 min rotating at room temperature, once with RIPA buffer, twice with 2 % SDS, once with RIPA 0.5 M NaCl, once with 1X LiCl buffer and twice with TE buffer. Chromatin was then purified and decrosslinked. Libraries were costructed using the NEB Next Ultra DNA Library prep kit (New England Biolabs) following manufacturer's instructions.Samples were indexed and 4 different samples sequenced together.