title: GSM4115966: GFP_PGCLC_noIAA_r2; Homo sapiens; ChIP-Seq
library_construction_protocol: The following samples were included in the analysis (one replicate per condition): 4i hESCs (cl11 no IAA), 4i+IAA hESCs (cl11+IAA) and hPGCLCs (cl11 and cl21 pooled; no IAA). For each ChIP, 2.5 million unsorted 4i hESCs or 4 million unsorted hPGCLCs (D4 EBs containing 60-70% PGCLCs as assessed by flow cytometry) were used. The cells were washed in PBS, filtered through 50 μm strainer and fixed in 1% formaldehyde at RT for 10 min. Formaldehyde was quenched by adding 450 μl of 10x glycine at RT for 10 min, followed by centrifugation at 500 g, 4 ºC for 5 minutes. The cells were then washed twice in ice-cold PBS with protease inhibitor cocktail (PIC) and the pellets were snap frozen on dry ice and stored at -80 ºC. ChIP was performed using SimpleChIP Enzymatic Chromatin IP Kit (with Magnetic Beads) from Cell Signalling Technology (CST) following manufacturer's recommendations with modifications. ChIP-seq libraries were prepared using KAPA HyperPrep Kit following the manufacturer's instructions. Briefly, the protocol contains the following steps: end-repair and A-tailing; sequencing adapter (index) ligation; product purification using AMPure beads; library amplification using KAPA real-time Library Amplification Kit (11 cycles were used for ChIP libraries and 7 for input libraries to achieve similar concentration range); product purification using AMPure beads. Libraries were then quantified by qPCR using NEBNExt Library Quant Kit for Illumina (NEB) on QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems). Fragment size distribution and the absence of adapter dimers were checked using Agilent TapeStation 2200 and High Sensitivity D1000 ScreenTape.
attributes.tag: GEO Accession
attributes.value: GSM4115966