title: GSM2806609: ATAC_Undecidualized sample 2; Homo sapiens; ATAC-seq
library_construction_protocol: ATAC-seq was performed as described (Buenrostro et al 2013), although with some modifications. Briefly, EnSCs were grown in 35 mm diameter tissue culture dishes. Confluent monolayers were washed with cold Dulbecco's Phosphate Buffered Saline (PBS) and then lysed using cold EZ lysis buffer (Sigma-Aldrich, UK). The cells were scraped and then transferred to chilled pre-labelled nuclease-free 1.5 ml micro-centrifuge tubes. Samples were vortexed, left on ice for 5 minutes, then pelleted in a fixed-angle refrigerated benchtop centrifuge. Supernatant was carefully removed and the pellet was washed once in EZ lysis buffer. The nuclear pellet was re-suspended in the transposase reaction mix, containing 25 μl Tagment DNA (TD) Buffer, 5 μl Tagment DNA Enzyme and 20 μl nuclease free water (Nextera DNA sample preparation kit, Illumina, UK).  The transposition reaction was carried out for either 45 or 90 min at 37 °C. Samples were purified using a Zymo DNA Clean and Concentrator-5 Purification kit, according to the manufacturer’s instructions. Briefly, DNA binding buffer was added to the 50 μl sample which was then mixed and transferred to the column. Samples were centrifuged at 17,000 × g for 30 secs at room temperature (RT). Flow-through was discarded and 200 μl DNA wash buffer added, and columns centrifuged as described above. The wash and centrifugation steps were repeated twice. After removing the residual liquid, 23 μl pre-warmed elution buffer was added to the columns. Samples were incubated for 2 min at RT, then centrifuged for 2 min to elute DNA. Following purification, 20 μl sample was added to a 0.2 mL PCR tube containing the following reagents (Nextera DNA sample preparation kit and Nextera index kit, Illumina): 5 μl index 1, 5 μl index 2, 15 μl Master mix (NPM), 5 μl Primer Cocktail (PPC). Amplification was performed in a Veriti 96 Well Thermal Cycler (Applied Biosystems, USA) using the following PCR conditions: 72 °C for 3 min, 98 °C for 30 secs then 15 cycles of 98 °C for 10 secs, 63 °C for 30 secs, 72 °C for 1 min. The libraries were purified using AMPure XP beads according to the Illumina Nextera kit recommended protocol. The amplified libraries were quantified by using Qubit HS DNA Assay on a Qubit 2.0 Fluorometer according to the manufacturer’s instructions. Library sizes were assessed on an Agilent Technologies 2100 Bioanalyzer using the High Sensitivity DNA chip.
attributes.tag: GEO Accession
attributes.value: GSM2806609