title: GSM7025130: Lmna Knockdown Dermal Fibroblast ATAC-Seq Rep 1; Homo sapiens; ATAC-seq
library_construction_protocol: 10-20,000 normal human dermal fibroblasts before and after Lmna knockdown and human embryonic stem cells were centrifuged at 500xg for 5 min at 4˚C in a fixed angle centrifuge. After removing the supernatant, the cell pellets were re-suspended in 50uL ATAC-Resuspension Buffer (RSB) (500uL 1M Tris-HCl, 100uL 5M NaCl, 150uL 1M MgCl2 in 49.25mL sterile water) with 0.1% NP40, 0.1% Tween-20, and 0.01% Digitonin. Samples were pipetted up and down three times to mix, incubated 3 minutes on ice, then diluted in 1mL of ice-cold ATAC-resuspension buffer (RSB) containing 0.1% Tween-20 and centrifuged at 500xg for 10 min at 4˚C to pellet the nuclei. The supernatants were carefully removed and the pellets were re-suspended in 25 µL of transposase reaction mix (12.5 µL 2x TD buffer, 1.25 µL TN5 transposase (Illumina) and 11.25 µL nuclease-free water). Transposase reactions were carried out by incubating samples at 37˚C for 30 min under mild agitation (300 rpm on a Thermo-mixer C, Eppendorf). Once the incubation was completed, sample tubes were placed on ice and the transposed DNA fragments from each sample were purified using a Qiagen MinElute PCR Purification Kit following manufacture's protocol. Purified DNA fragments were then amplified for 13 cycles using barcoded PCR primers and NEB Next High Fidelity 2x PCR Master Mix (New England Bio Labs) on a thermal cycler. Double concentrated Ampure beads were used to purify transposed DNA amplicons. The molarity of each DNA library was determined (Agilent 2100 Bioanalyzer), pooled into a single tube and sequenced on a NextSeq (Illumina) using 76-bp paired-end reads.