title: GSM3984861: Sonicated rep1; Homo sapiens; ATAC-seq
library_construction_protocol: We conducted ATAC-seq as described in Buenrostro et al. (Curr. Protoc. Mol. Biol. 2015) using a home-made Tn5 that we synthesized as described in Picelli et al. (Genome Res. 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, Genome Res. 2012) and eluted in 22 uL of TTE8 buffer.
identifiers.namespace: GEO
identifiers.id: GSM3984861