title: GSM3731833: ATACMe_THP1_0hrPMA_rep1; Homo sapiens; OTHER
library_construction_protocol: THP1 cell suspension volume corresponding to 2E5 cells was pelleted in a 1.5mL tube at 4°C, 500 R.C.F for 5 minutes. Supernatant was aspirated and the pellet resuspended in 150µL cold ATAC lysis buffer (10mM Tris-HCl pH = 7.4, 10mM NaCl, 3mM MgCl2, 0.1% IGEPAL-630). This suspension was agitated by pipetting up/down with a 200µL micropipette tip 8 times. Subsequently, this suspension was pelleted at 4°C, 500 R.C.F for 10 minutes. Supernatant was discarded and pellet immediately resuspended in 190µL transposition reaction mix (10mM Tris-HCl pH=7.5, 5mM MgCl2, 10% Dimethylformamide) by pipetting up/down with a 200µL micropipette tip 3 times. After resuspension 10µL of Tn5 transposase pre-assembled with methylated adapters was added. Tube was flicked gently to mix all components and incubated at 37°C, 30 minutes, 700RPM in an Eppendorf Thermomixer. Immediately after the 30-minute incubation ATAC reactions were terminated by adding 1mL Zymo DNA binding buffer and vortexing. This binding buffer and ATAC reaction mixture was purified according to manufacturer instruction in the Zymo DNA Clean and Concentrator-5 kit. Final elution was in 12.5µL nuclease free H2O. After elution gap repair reactions were assembled and incubated as described in “Wang, Q. et al. Tagmentation-based whole-genome bisulfite sequencing. Nature Protocols 8, 2022-2032 (2013)“ with slight modification (11µL ATAC-Me DNA eluate, 2µL 10µM Tn5mC-Repl01 oligo, 2µL 10x ampligase buffer, 2µL dNTPs 2.5mM each)17. 2µL of the gap repair reaction was reserved for a test PCR amplification to confirm successful ATAC nucleosomal laddering. Subsequently, 2µL of 250mM EDTA (pH=8.0) was added to stop the reaction. Gap repaired ATAC-Me material was bisulfite converted according to manufacturer instructions using the EZ DNA Methylation-Lightning Kit (Zymo). Final elution was in 22µL of M-elution buffer supplied by the kit. Gap repaired, bisulfite converted ATAC-Me DNA reactions were PCR amplified with 12 cycles of amplification using HiFi HotStart Uracil + Ready Mix (KAPA) and barcoded primers. Post-amplification PCR reactions were cleaned and concentrated with a DNA Clean and Concentrator-5 column kit (Zymo). Elution was in 22µL nuclease free H2O. Preliminary library analysis for concentration and size distribution was performed using an Agilent 2200 TapeStation with a D5000 screentape.
attributes.tag: GEO Accession
attributes.value: GSM3731833