GSM5592292: atac.no.crush.rep6; Mus musculus; ATAC-seq
Samples were dissociated in papain, followed by FACS sorting. For ATAC-seq, 50,000 - 100,000 sorted RGCs were lysed in 100 µL ice-cold Resuspension Buffer (RSB, 10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, pH=7.4) with 0.1% NP40, 0.1% Tween- 20, and 0.01% Digitonin. Cell lysis were washed with 1 ml of cold ATAC-RSB containing 0.1% Tween-20 but NO NP40 or digitonin, and centrifuged at 4 °C for 10 minutes at 500 x g. Pelleted nuclei were resuspended gently in 50 µL transposition mix (25 ul 2x TD buffer, 2.5 ul transposase (100nM final), 16.5 ul PBS, 0.5 ul 1% digitonin, 0.5 ul 10% Tween-20, 5 ul H2O) and incubated for 30 minutes at 37 °C. DNA was cleaned using Zymo DNA Clean and Concentrator-5 Kit. Purified DNA was PCR amplified 5 cycles in a 50 µL reaction with Illumina Nextera adaptors using NEBNext High Fidelity 2x Master Mix. To determine the number of additional cycles to amplify the libraries, a side qPCR reaction was performed using 5 µL (10%) of the pre-amplified PCR. We used the Ct value of the qPCR at ¼ maximum fluorescence as the number of additional cycles. Final PCR products were cleaned using Zymo DNA Clean and Concentrator-5 Kit, and quantified by the KAPA Library Quantification kit prior to pooling and sequencing.