GSM4626191: NHBE_1-2; Homo sapiens; ATAC-seq
Omni-ATAC-seq library preparation was performed as previously described (PMCID: PMC5623106). Airway epithelial cells and cultured lung fibroblasts were grown to ~70% confluency, harvested by trypsinization and washed in cold PBS. Aliquots of 50,000 cells were centrifuged for 5 min at 4°C and 500 × g then resuspended in ATAC-resuspension buffer (RSB) containing NP40, Tween-20, and digitonin for 3 min on ice. ATII cells were aliquoted immediately after FACS sorting and similarly resuspended in RSB. Cells were washed with RSB containing Tween-20 only, centrifuged, and resuspended in transposition mixture containing PBS, Tween-20, digitonin and transposase enzyme (Nextera). Fast-ATAC-seq of 16HBE cells was performed as previously described (PMCID: PMC5042844), wherein the RSB lysis step is omitted and the cells were immediately resuspended in transposase mixture containing only digitonin as detergent. The transposition reaction was incubated at 37°C with shaking at 1000 RPM for 30 min. After transposition, DNA was purified with the MinElute Reaction Clean-up Kit (QIAGEN) and eluted in 20 μL elution buffer. The libraries were pre-amplified as previously described for 5 cycles and an analytical qPCR was performed to determine the additional number of amplification cycles required to ensure sufficient material and limit PCR duplication. The final PCR amplification was performed, and libraries were again purified and eluted in 20 uL H20. Prior to sequencing the libraries were quantified by Agilent D1000 ScreenTape on the Agilent 2200 TapeStation system and SYBR qPCR to assess functional concentration. Transposase treatment and library construction was performed using the Nextera DNA Library Preparation Kit (Illumina, FC-121-1030) and custom ordered Nextera PCR primers as described (PMCID: PMC3959825).
GEO Accession
GSM4626191