GSM5454513: Gabra3_3WT_Retina_ATAC; Mus musculus; ATAC-seq
Flash-frozen tissue was thawed by addition of 700ul of lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40), homogenized by trituration 10x with a p1000 pipet set to 500ul, dounced in an ice cold RNase-free 2mL glass dounce 10x with a loose pestle and 10x with tight pestle, and transferred back to original 1.5mL LoBind tube (Eppendorf). Dounce was washed with an additional 700ul ice cold lysis buffer and buffer was transferred to tube with sample for a final volume of 1.4mL. Sample was then centrifuged in a microcentrifuge at 4C for 10min at 500 rcf. The pellet was re-eluted in 1.4mL ice cold lysis buffer, transferred to a pre-chilled dounce, homogenized again 12x with tight pestle, and transferred back to 1.5mL LoBind tube. Nuclei were counted on a hemocytometer and the volume of lysis buffer/nuclei suspension needed for 20,000 nuclei was aliquoted into a separate 1.5mL LoBind tube. Samples were centrifuged along with tube containing remaining nuclei (for nuclear localized RNA) at 4C for 5min at 500g (RCF). The supernatant was removed from samples and remaining nuclei. Subsequent ATAC libraries were generated with 20,000 nuclei per sample using the Nextera DNA library prep kit (FC-121-1030; Illumina, San Diego, CA USA). ATAC-Seq libraries were prepared as described above and sequenced on the Illumina NextSeq 500 platform with 75 bp single-end reads.