GSM7910908: Alim Pre-Diapause ATACseq Rep2; Austrofundulus limnaeus; ATAC-seq
To generate nuclei-suspension for ATAC-seq libraries, snap frozen embryo samples (~10 embryos per sample) were thawed for 1 minute and resuspended at 4oC in 200ml EZ-lysis buffer (Sigma Aldritch No. 3408). Samples were then transferred to 250 ml mini-douncers (DWK (Kimble) 885300-0000) and dounced 25 times with pestle A and B respectively. After a 2 minute incubation following douncing, samples were spun at 500g for 5 minutes to precipitate nuclei, and the EZ-lysis supernatant was removed. Nuclei were then resuspended in 250ml PBS (ThermoFisher No. AM9624) and an aliquot of 5µl of nuclei was incubated with 5ml of 0.4% trypan blue stain (ThermoFisher No. 15250061) for counting the total intact nuclei counts. Samples of ~25,000 nuclei were then suspended in a Tn5 transposition mix (65ml of tagmentation DNA buffer (Illumina No. 20034197), 63ml of nuclease-free water, and 2.5ml of tagmentation DNA enzyme I (e.g Tn5 transposase) (Illumina No. 20034197) for 20 minutes at 37oC. Following incubation, the mix was purified using the Qiagen mini-elute kit (Qiagen No. 28206) to isolate tagmented DNA. PCR amplification and subsequent qPCR monitoring was performed as described in the original ATAC-seq protocol (~14-18 cycles of PCR). Amplified DNA from the PCR reaction was purified using the Qiagen mini-elute kit (Qiagen No. 28206), as recommended by the manufacturer. Samples were subsequently pooled and sequenced using next-generation short-read sequencing on an Illumina NEXTseq 550.