GSM2858068: XM001-PP Input Lane2 GGCTAC; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Input control
Antigen
Input control
Cell type
Cell type Class
Pluripotent stem cell
Cell type
iPSC derived pancreatic cells
NA
NA
Attributes by original data submitter
Sample
source_name
iPS derived pancreatic precursor cells
cell type
iPS derived pancreatic precursor cells
chip target
none (Input)
lane
Lane2
antibody
none
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
XM001 PP cells (2 × 10^6 cells) were cross-linked in 1% formaldehyde in culture medium for 10 min at room temperature. The cross-linking reaction was stopped by the addition of glycine to a final concentration of 125 mM. For chromatin fragmentation, cells were resuspended in lysis buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.5% SDS) and sonicated in a Covaris S220 sonicator with a duty cycle of 2%, a peak incident power of 105 W and 200 cycles per burst for 20 min. The fragmented chromatin was diluted 1:5 in IP-Buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% SDS, 0.1% Na-Desoxycholate, 140 mM NaCl, H2O, Protease Inhibitors) and directly used for immunoprecipitation. For PDX1 ChIP, 60% of the chromatin (equivalent of 1.2 × 10^6 cells) was processed in two parallel ChIPs using magnetic beads, preloaded with 3 µl goat anti PDX1 antibody (kindly provided by C. Wright), for each ChIP. For H3K27ac ChIP, 40% of the chromatin (equivalent 0.4 × 10^6 cells) was processed in three parallel IPs using magnetic beads, preloaded with 3 µg anti H3K27ac antibody (Diagenode, pAb-174-050). For all ChIPs, antibody incubation was performed at 4 °C for 5 h. Beads were then washed 5 times using (1.) IP-Buffer, (2.) Washing-Buffer 1 (500 mM NaCl, 50 mM Tris-HCl (pH 8.0), 0.1% SDS, 1% NP-40), (3.) Washing-Buffer 2 (250 mM LiCl, 50 mM Tris-HCl (pH 8.0), 0.5% Na-Deoxycholate, 1% NP-40) and (4. & 5.) Washing-Buffer 3 (10 mM Tris-HCl (pH 8.0), 10 mM EDTA). Subsequently, protein-DNA complexes were eluted from the beads in Elution-Buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 1% SDS) at 65 °C for 20 min. Cross-links were reversed at 65 °C overnight and DNA was purified by ethanol precipitation. Libraries were constructed using the MicroPlex kit (Diagenode, C05010010). Library construction, purification and size selection using AMPureXP (Beckman Coulter) were performed according to the MicroPlex kit instruction manual (version 4 / 03.09.14)