title: GSM5026635: Ch12 Klf16 Maz Sp1 Zbtb7a KO S1R1; Mus musculus; ATAC-seq
library_construction_protocol: ATAC-seq: To prepare nuclei for tagmentation, 100,000 cells were centrifuged at 500 × g for 5 minutes, then washed with 50 μL of cold 1x PBS and centrifugated at 500 × g for 5 minutes. Cells were lysed using cold lysis buffer (10 mM Tris-Cl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1% IGEPAL CA-630). Immediately after lysis, nuclei were centrifuged at 500 × g for 10 minutes using a refrigerated centrifuge. Immediately following the nuclei prep, the pellets were resuspended in the transposase reaction mix (25 μL 2x TD buffer, 2.5 μL Transposase (Illumina) and 22.5 μL of nuclease free water). The transposition reaction was carried out for 30 minutes at 37 °C. Directly following transposition the sample was purified using a Zymo Clean and Concentrator kit. After purification, library fragments were amplified using 1x NEBnext PCR master mix and 1.25 μM of custom Nextera PCR primers, using the following PCR conditions: 72°C for 5 minutes, 98°C for 30 seconds, followed by 4 cycles at 98°C for 10 seconds, 63°C for 30 seconds and 72°C for 1 minute. To reduce GC and size bias in our PCR, the PCR reaction was monitored using qPCR in order to stop amplification prior to saturation. A 5 μl aliquot of the PCR reaction was mixed with 10 μl of the PCR cocktail with Sybr Green at a final concentration of 0.6x, and a 20-cycles qPCR was run to determine the additional number of cycles needed for the remaining 45 μL reaction. The libraries were then purified using the Zymo Clean and Concentrator kit. ATAC-seq: DNA was prepared for single-end Illumina sequencing. 50 cycles of sequencing data were acquired on the Illumina NovaSeq 6000.