GSM1697141: Input DNA H3.3WT; Mus musculus; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Input control
Antigen
Input control
Cell type
Cell type Class
Embryonic fibroblast
Cell type
C3H/10T1/2
NA
NA
Attributes by original data submitter
Sample
source_name
C3H10T1/2 cells & S2 cells
cell line
C3H10T1/2
cell type
C3H embryo-derived mesenchymal progenitor cells
genotype
Stably expressing FLAG-HA-tagged wildtype H3.3
passages
Passage 10-15
spike-in
soluble chromatin from Drosophila melanogaster S2 cells derived from a primary culture of 20–24 hours old, late stage embryos (equivalent to 5% of the mouse cell chromatin)
chip antibody
none
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
~2x10^7 C3H10T1/2 cells were lysed using digesting buffer (50 mM Tris-HCl pH 7.6, 1 mM CaCl2 and 0.2% Triton X-100) and digested with micrococcal nuclease to obtain mono-nucleosomes. The chromatin were then dialyzed into RIPA buffer (10 mM Tris pH 7.6, 1 mM EDTA, 0.1% SDS, 0.1% Na-Deoxycholate, 1% Triton X-100) for 2 h at 4°C. After centrifugation, soluble chromatin was spiked-in with soluble chromatin from Drosophila S2 cells that was similarly prepared and equivalent to 5% of the mouse cell chromatin. The mixed soluble chromatin was incubated with αH3K36me3 (Active Motif, 61101), αH3K36me2 (Millipore, 07-369-I) or αH3K27me3 (Cell Signaling Tech, 9733) antibody bound to 75 μl protein A or protein G Dynal magnetic beads (Invitrogen) and incubated overnight at 4°C, with 5% kept as input DNA. Magnetic beads were washed with RIPA buffer and LiCl buffer (0.25 M LiCl, 0.5% NP40, 0.5% Na-Deoxycholate) and chromatin was eluted. ChIP DNA was treated with Proteinase K (Roche) and recovered using Qiagen PCR purification kit. libraries were prepared according to the Illumina TruSeq protocol