alias: GSM4271526
title: GSM4271526: 8-cell_amanitin_rep1_ATACseq; Bos taurus; ATAC-seq
library_construction_protocol: Oocytes and embryos were collected for ATAC-seq library preparation from three separate collections per developmental stage. Embryos intended for collection at the 2-cell, 4-cell, or 8-cell stages were divided into two groups, one of which was supplemented with α-amanitin, and cultured simultaneously. Germinal vesicle-stage oocytes were collected for ATAC-seq prior to maturation. Preimplantation embryos were collected at the 2-cell (30-32 h post-insemination), 4-cell (2 dpi), 8-cell (3 dpi), and morula stages (5 dpi). Oocytes or embryos (a minimum of 500 cells) were treated with pronase (10 mg/ml) to completely remove the zona pellucida and washed with SOF-Hepes on a warming plate. Cells were then transferred to 1 ml cold SOF-Hepes, and centrifuged at 500 rcf, 4°C, for 5 min. Morulae were subjected to additional vortexing for 3 min in cold ATAC-seq lysis buffer (10 mM Tris-Cl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1% IGEPAL CA-630). Cell pellets were then resuspended in 1 ml cold ATAC-seq cell lysis buffer and centrifuged at 500 rcf, 4°C, for 10 min, and supernatant was aspirated to isolate nuclear pellets. Nuclear pellets were resuspended in 50 μl transposition reaction mix (25 μl TD buffer (Nextera DNA Library Prep Kit, Illumina), 2.5 μl TDE1 enzyme (Nextera DNA Library Prep Kit, Illumina), 22.5 μl nuclease-free H2O) and incubated for 60 min at 37°C, shaking at 300 rpm. Transposed DNA was purified with the MinElute PCR purification kit (Qiagen, Hilden, Germany) and eluted in 10 μl buffer EB. Libraries were then PCR amplified. 50 μl reactions (25 μl SsoFast Evagreen supermix with low ROX (Bio-Rad, Hercules, CA), 0.6 μl 25 μM custom Nextera PCR primer 1, 0.6 μl 25 μM custom Nextera PCR primer 2 (for a list of primers, see Buenrostro et al (2015)), 13 μl nuclease-free H2O, and 10 μl eluted DNA) were cycled as follows: (1) 72°C for 5 min, 98°C for 30 s, then (2) thermocycled at 98°C for 10 s, 63°C for 30 s and 72°C for 1 min. Libraries from GV oocytes, 2-cell, and 4-cell embryos were thermocycled for 13 cycles, and 8-cell and morulae libraries were thermocycled for 11 cycles. PCR-amplified libraries were again purified with the MinElute PCR purification kit and eluted in 10 μl buffer EB. The sub-nucleosomal length fraction of each library (150-250 bp) was size selected using the PippinHT system (Sage Science, Beverly, MA) with 3% agarose cassettes.
identifiers.namespace: GEO
identifiers.id: GSM4271526