title: GSM5538153: ES INPUT Rep2; Homo sapiens; ChIP-Seq
library_construction_protocol: For RNA-Seq experiments, Total RNA was extracted using the Nucleospin RNA Extraction kit.For ChIP-Seq experiments, after differentiation to the desired stage, cells were dual crosslinked in plate with 1.5mM EGS and 1% formaldehyde. The fixation was then quenched with 125mM glycine. Cells were then washed twice and scraped in ice-cold PBS and resuspended in sonication buffer.For ATAC-Seq experiments, 50,000 cells were collected following differentiation to the desired stage, and lysed in 50ul of ATAC-lysis buffer (10mM Tris-HCl, pH 7.4. 10mM NaCl, 3mM MgCl2, 0.1% NP-40) to obtain a crude nuclei prep For RNA-Seq experiments, 300ng of total RNA was poly-A selected and reverse transcribed using Illumina's TruSeq stranded mRNA library preparation kit (Illumina; 20020595). For ChIP-Seq experiments, DNA libraries were prepared using 1-5 ng of starting material using the SMARTer ThruPLEX DNA-Seq kit (Takara; R400674) according to manufacturer's instructions. For ATAC-Seq, the eluted DNA was amplified in a reaction with 25ul NEBNext High-Fidelity 2x PCR Mastermix (NEB; M0541L) and custom 25um Nextera PCR Primers (Ad1_noMx universal primer, 0.5um Ad2.x indexing primer). PCR was performed as follows: 1 cycle of 72C for 5min, 98C for 30s, 5 cycles of 98C for 10s, 63C for 30s, 72C for 1min. 5ul of the amplified DNA was then used to perform qPCR to determine the optimal number of additional cycles to prevent amplification saturation of DNA libraries. In all cases, either 4 or 5 additional cycles of PCR was performed at: 98C for 10s, 63C for 30s and 72C for 1 min. Double size selection of amplified libraries (0.5x – left sided, 1.8x – right sided) was performed using AMPure XP beads ChIP-Seq, RNA-Seq and ATAC-Seq