title: GSM4800907: SMARCA4KO_SMARCA2dTAG_DMSO_H3K27ac; Homo sapiens; ChIP-Seq
library_construction_protocol: ATAC-seq was performed according to Buenrostro et al. with small adaptations (Buenrostro et al., 2015). In brief, 50,000 cells (1:2 drosophila S2 cells : HAP1 cells) were resuspended in 25 µl transposase reaction mix (0.02% digitonin (Promega G9441), 1x TD buffer, 0.08% TDE1 (Nextera DNA Library Preparation Kit, Illumina, FC-121-1031)) and incubated for 30 min, 37°C, 300 rpm. Then DNA was purified using MinElute kit (Qiagen, 28004) and eluted in 11 µl elution buffer. On-plate ATAC-seq was performed as described for ATAC-seq, but without Drosophila spike-ins and performing transposase reaction on the plate. ChIP-seq and RNA-seq was performed as described in Schick et al., 2019. Pro-seq was performed as described in Jaeger et al., 2020. ATAC-seq: 1 µl was used to determine cycle number for PCR using a qPCR approach. The remaining 10 µl were complemented with 1x NEBnext High-Fidelity PCR master mix (New England BioLabs, M0541), 1.25 µM index primer 1 and 1.25 µM index primer containing a barcode. PCR was performed: 5 min 72°C, 30 sec 98°C, X cycles of 10 sec 98°C + 30 sec 63°C + 1 min 72°C, 1 min 72°C and then cleaned-up using Agencourt AMPure XP beads (Beckman Coulter, A63880). The libraries were checked on a Bioanalyzer instrument using High Sensitivity DNA Chips (Agilent), quantified using Qubit dsDNA HS Assay kit (Thermo Fisher Scientific, Q32854) and pooled. Libraries were sequenced using the Illumina HiSeq3000/4000 platform with the 75 bp paired-end configuration.   ChIP-seq: NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs E7645) was used for the library preparation as described by manufacturer protocol. The quality control of the ChIP samples was confirmed prior to sequencing on Bioanalyzer (Agilent Cat no. 5067-4626). Samples were sequenced on a HiSeq 3000/4000 platform using 50 bp single read configuration.  RNA-seq: RNA-seq libraries were prepared with the TruSeq Stranded mRNA LT sample preparation kit (Illumina, San Diego, CA, USA) using Sciclone and Zephyr liquid handling workstations (PerkinElmer, Waltham, MA, USA) for pre- and post-PCR steps, respectively. Library concentrations were quantified with the Qubit 2.0 Fluorometric Quantitation system (Life Technologies, Carlsbad, CA, USA) and the size distribution was assessed using the Experion Automated Electrophoresis System (Bio-Rad, Hercules, CA, USA). For sequencing, samples were diluted and pooled into NGS libraries in equimolar amounts. Expression profiling libraries were sequenced on HiSeq 3000/4000 instruments (Illumina, San Diego, CA, USA) in 50-base-pair, single-end mode.  PRO-seq: Finally, RNA was reverse transcribed using RP1 primer with Superscript III and cDNA subjected to a 4x-dilution series test PCR to determine the optimal PCR cycle number for final sequencing library amplification. Full scale amplification with RPI-x indexing primers was then performed in 2x 50 µl reactions per sample for a total of 11-19 cycles. DNA was ethanol precipitated and subjected to 2x 1.1:1 AMPure XP bead left-side size selection according to manufacturer instructions. DNA concentration was determined using the Qubit dsDNA Hs kit and library size distribution was measured with Bioanalyzer. Libraries were pooled to equal molar ratios and sequenced on a HiSeq 3000/4000 system in 50SR mode, yielding ~60 mio reads per sample.
attributes.tag: GEO Accession
attributes.value: GSM4800907