GSM3577161: NANOG Chip-seq in WIBR2 cell line, maintained in HENSM conditions [NANOG HENSM WIBR2]; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
NANOG
Cell type
Cell type Class
Pluripotent stem cell
Cell type
hESC WIBR2
NA
NA
Attributes by original data submitter
Sample
source_name
hESCs, WIBR2 cell line
cell line
WIBR2, 29-8 cell line
culture conditions
HENSM
target protein
NANOG
matched control file
WCE_HENSM_WIBR2_10
antibody
R&D Systems AF1997
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were crosslinked in formaldehyde (1% final concentration, 10 min at room temperature), and then quenched with glycine (5 min at room temperature). Fixated detailed in the table below were then lysed in 50 mM HEPES KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40 alternative, 0.25% Triton supplemented with protease inhibitor at 4 °C (Roche, 04693159001) for 10 min, and later centrifuged at 950g for 10 min. Supp was discarded and pellet was resuspended in RIPA-1 (0.2% SDS, 1 mM EDTA, 0.1% DOC, 140 mM NaCl and 10 mM Tris-HCl) with protease inhibitor. Cells were then fragmented with a Branson Sonifier (model S-450D) at −4 °C to size ranges between 200 and 800 bp and centrifugation at max speed for 10 min. Supp lysate was extracted and diluted with RIPA 2-3 fold (0.1% SDS, 1 mM EDTA, 0.1% DOC, Triton 1%, 140 mM NaCl and 10 mM Tris-HCl). Small amount of lysate were saved for whole cell extract at this point. Antibody was pre-bound by incubating with Protein-G Dynabeads (Invitrogen 10004D) in blocking buffer (PBS supplemented with 0.5% TWEEN and 0.5% BSA) for 1 h at room temperature. Washed beads were added to the lysate for incubation as detailed in the table below. Samples were washed five times with RIPA buffer, twice with RIPA buffer supplemented with 500 mM NaCl, twice with LiCl buffer (10 mM TE, 250mM LiCl, 0.5% NP-40, 0.5% DOC), once with TE (10Mm Tris-HCl pH 8.0, 1mM EDTA), and then eluted in 0.5% SDS, 300 mM NaCl, 5 mM EDTA, 10 mM Tris HCl pH 8.0. Eluate was incubated treated sequentially with RNaseA (Roche, 11119915001) for 30 min in 37°C and proteinase K (NEB, P8102S) for 2 h in 37°C and de-crosslinked in 65 °C for 8 h. DNA was purified with The Agencourt AMPure XP system (Beckman Coulter Genomics, A63881) 120ul SPRI AMPure XP beads (Agencourt) were added to the reverse-crosslinked samples, pipette-mixed 15 times and incubated for 2 minutes. Supernatant were separated from the beads using a magnet for 4 minutes. Beads were washed on the magnet with 70% ethanol and then air dried for 4 minutes. The DNA was eluted in 40 ul EB buffer (10 mM Tris-HCl pH 8.0) by pipette mixing 25 times. For the remainder of the library construction process (DNA end-repair, A-base addition, adaptor ligation and enrichment) a general SPRI cleanup involves addition of buffer containing 20% PEG and 2.5 M NaCl to the DNA reaction products (without moving them from their original well position). After thorough mixing and a 2-minute incubation at room temperature, plates are transferred to a magnet plate, incubated for 4 minutes and supernatant removed. Beads are then washed on the magnet with 150ul 70% ethanol and then air dried for 4 minutes. The DNA is eluted with 40ul of EB buffer by pipette mixing 25 times. Reagent kits are prepared in advance for all enzymatic steps (New England Biolabs). The DNA end-repair was performed by adding 27 µl of a master mix (17 µl master mix (5 ul T4 buffer, 5ul BSA-1mg/ml, 5ul ATP-10mM -2ul dNTPs 10 mM), 5 ul T4 PNK enzyme, 5 µl T4 polymerase (3 units) to each well. Samples were incubated in a thermal cycler at 12C for 15 min, 25C for 15 min, and finally cooled to 4C. The SPRI bead clean up method was used to purify the product (147 µl of 20% PEG, 2.5 M NaCl was added to each sample and eluted in 40 µl EB). The A-base addition was performed by adding 20 µl master mix (17 µl A-base add mix, 3 µl Klenow (3'->5' exonuclease) to each well and incubated at 37C for 30 min. in a thermal cycler. SPRI bead clean up method was used to purify the product (132 µl of 20% PEG, 2.5 M NaCl was added to each sample and eluted in 19 µl EB). Adaptor ligation was performed by adding 34 µl of a master mix (29 µl 2x DNA ligase buffer, 5 µl DNA ligase) to each well. Finally 5 µl PE Indexed oligo adaptors (0.75 uM ) was added to each well and samples were incubated 25C for 15 min in a thermal cycler. SPRI bead clean up with size selection was used to purify the ligated products (15.5 µl of 20% PEG, 2.5 M NaCl was added to each sample and eluted in 40 µl EB). Finally, enrichment PCR was performed by adding 10 µl of a master mix (2 µl Forward/Reverse Index Primer, 0.5 µl dNTP mix, 5 µl 10x Pfu Ultra Buffer, 1 µl Pfu Ultra-II Fusion, 1.5 µl Nuclease free water) to each well. Plate was transferred to a thermal cycler and ran a Pfu amplification program at 95C for 2 min, 16 cycles of: 95C for 30 sec, 55C for 30 sec, 72C for 60 sec, and finally 72C for 10 min. The final SPRI clean up coupled to size selection was performed (35 µl SPRI beads was added to each sample and eluted in 40 µl).