title: GSM7093940: D0 rep 1 control; Homo sapiens; ATAC-seq
library_construction_protocol: Cells were lysed by adding 20 µL/well of ATAC suspension buffer (RSB) (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2) containing 0.1% NP40 (Sigma Aldrich), 0. 1% Tween-20 (Sigma Aldrich) and 0.01% Digitonin (Promega), and incubated for 3 min on ice, then washed with 0.1 mL of cold ATAC-RSB containing 0.1% Tween-20 without NP-40 and digitonin. The extracted nuclei were centrifuged at 500 g for 10 min at 4°C. DNA libraries for ATAC-Seq were prepared at Institute Cochin's GENOM'IC facility, from transposed DNA using the Nextera® DNA kit from Illumina, following the final amplification by PCR step instructions. Reaction tubes contained 15µL Nextera PCR Master Mix, 5µL Illumina PCR Primer Cocktail, 5µL Index Primer i7, 5µL Index Primer i5, 10µL H2Oand 10µL of Transposed DNA. A PCR was performed with the following parameters : 3' @72°C, 30'' @98°C, and eight cycles of 10'' @98°C, 30'' @60°C, 2' @72°C. PCR products were purified using AMPureXP™ beads from Agencourt at a 1X ratio. Final libraries quality was assessed on a DNA High Sensitivity™ chip on the Bioanalyzer 2100 from Agilent, and concentration was measured using the Qubit dsDNA HS Assay™ kit from Thermo Fisher Scientific (average yield was 12.1 ng/µL). Molarity from each library was calculated with the following formula: Qubit measure * 1000000 / ((average peak size in bp * 607.4)+157.9).  Each library was diluted to reach molarity of 10nM, and a final pool was set at 2nM prior to sequencing.  Sequencing of the ATAC-Seq libraries was performed on a NextSeq™ 500 from Illumina (GENOM'IC). Sequencer parameters were set as following : read 1 = 76 cycles, index 1 = 8 cycles, index 2 = 8 cycles, read 2 = 76 cycles (using a NextSeq 500 High Output 150 cycles reagent kit from Illumina). Internal PhiX control was added in the library pool to reach 1% of the total reads. The sequenced pool was diluted to a 2pM concentration and loaded in the sequencer.