title: GSM3984863: Sonicated rep3; Homo sapiens; ATAC-seq
library_construction_protocol: We conducted ATAC-seq as described in Buenrostro et al. (2015) using a home-made Tn5 that we synthesized as described in Picelli et al. (2014). For each replicate we incubated 250,000 cells with three different concentrations of enzyme (0.2X, 1X, and 5X; 1X corresponds to 2.5 uL of Tn5 that carry 5-methylC-MEDS-A oligos) at 37℃ for 30 minutes in a 50 uL reaction. We column-purified the tagmented DNA using the Zymo DNA Clean & Concentrator-5 kit (Zymo Research, Irvine, CA), and sonicated to ~350 bp using the Covaris M220 sonicator (peak incident power - 50W; duty factor - 20%; cycles per burst - 200; treatment time - 60 sec). We constructed Illumina sequencing library using the ACCEL-NGS Methyl-seq DNA Library kit (Swift Biosciences #DL-ILMMS-12; revision 160106) with the following exceptions to the manufacturer's protocol: 1) We skipped “Ligation” and “Post-ligation SPRI” steps (pg. 10-11), as 5' end had already been tagged by Tn5 during the transposition step. Accordingly, we eluted DNA with 20 uL of TTE8 (10 mM Tris-HCl, 0.1 mM EDTA, 0.05% Tween-20, pH 8) for Post-Extension SPRI step (pg. 10), instead of 15 uL, to make up the difference in volume before proceeding to the “Indexing PCR” step (pg. 11); 2) We used 2:1 beads:sample ratio for “Post-Extension SPRI” step (pg. 10) and 1.8:1 beads:sample ratio for “Post-PCR SPRI” step (pg. 12); and 3) For indexing PCR, we used the Kitzman lab custom primers (barcode plate #5) to prime the P5 end and the “IndexD7XX” primers (Swift Biosciences #DI-ILMMS-48) to prime the P7 end. We PCR-amplified a total of 14 cycles for 0.2X, 1X samples and 16 cycles for 5X samples until amplification curve reached its mid-log phase (⅓ to ½ of max signal), and then purified the products using SPRI beads prepared as in (Rohland and Reich, 2012) and eluted in 22 uL of TTE8 buffer.
identifiers.namespace: GEO
identifiers.id: GSM3984863