GSM4037461: Chip-seq Pol2 in Chaserrb/b mESCs; Mus musculus; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Unclassified
Antigen
Unclassified
Cell type
Cell type Class
Pluripotent stem cell
Cell type
ES cells
NA
NA
Attributes by original data submitter
Sample
source_name
mouse embryonic stem cells
cell type
mouse embryonic stem cells
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
1x107mEFs were crosslinked with formaldehyde at 1% final concentration, 10 min at room temperature, and then quenched with 125mM glycine for 5 min at room temperature. Cells were centrifuged at 5min, 2000rpm at 4˚, and washed twice with PBS supplemented with PIC (protease inhibitors cocktail). Each pellet was then lysed in 1mL lysis buffer (5 mM PIPES pH 8.0 85 mM KCl, Igepal (10 µl/ml), and PIC) and incubated 15min at 4˚, then centrifuged for 5min at 15000rpm at 4˚. Pellets were resuspended in 200µl nuclei lysis buffer (50 mM Tris-Cl pH 8.1, 10 mM EDTA, 1% SDS, and PIC) for 30min at 4˚. Lysates were then sonicated (Bioruptor, #B01020001) for 12 cycles of 30s ON, 30s OFF. Pol2 antibody (5µg, anti-Rbp1 NTD (D8L4Y), #14958) was bound to A/G magnetic beads in 1mL binding/washing buffer (PBS supplemented with 0.5% TWEEN and 0.5% BSA) for 1 h at room temperature. Chromatin lysate was then diluted x9 volume with IP dilution buffer (50mM Tris-HCl pH 7.4, 150mM NaCl, 1% Igepal, 0.25% Deoxycholic acid, 1mM EDTA pH 8.0), then washed coupled beads were added to the chromatin lysate and left for slow rotation O.N at 4˚. Beads were handled as described68 .Briefly, beads were washed 5 times with RIPA buffer(10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 140 mM NaCl, 1% Triton X-100, 0.1% SDS and 0.1% Na-DOC), twice with RIPA-500 buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 500 mM NaCl, 1% Triton X-100, 0.1% SDS and 0.1% Na-DOC), twice with LiCl buffer(10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 250 mM LiCl, 0.5% NP-40 and 0.5% Na-DOC), once with TE buffer (10 mM Tris-HCl pH 8.0 with 1 mM EDTA pH 8.0). Beads were eluted with direct elution buffer (10 mM Tris-HCl pH 8.0, 5 mM EDTA pH 8.0, 300 mM NaCl and 0.5% SDS) at room temperature. Eluate was incubated with RNaseA (Homemade) for 30 min at 37˚,next with proteinase K for 2 h in 37°C and lastly O.N at 65˚. DNA was purified with The Agencourt AMPure XP system (Beckman Coulter Genomics, A63881). Libraries were constructed as previously described68, sequenced with paired-end sequencing on Illumina NextSeq 500.