GSM6005313: ADC9010, 2B; Homo sapiens; ATAC-seq
GSM6005313
Tissue samples were finely chopped on ice and homogenized in an Accumax solution (Sigma-Aldrich) using a Dounce homogenizer. The samples were then passed through a 100um cell strainer followed by a 40um cell strainer. The cells were pelleted in a cold centrifuge at 2,000 rpm and washed with cold PBS twice. We resuspended the pellet in 125uL of ATAC-Resuspension Buffer (RSB) containing 0.1% NP-40, 0.1% Tween-20 and 0.01% Digitonin, and incubated on ice for 3 minutes. We washed the lysis with 5ml ATAC wash buffer (10mM Tris-HCL, 10mM NaCl, 3mM MgCl2, 1% BSA, 0.1% Tween-20) and counted nuclei using trypan blue (1:9). We kept 50K nuclei from each sample, and centrifuged at 500 RCF (10 minutes, 4C). Later samples were using an improved protocol that includes sorting: These samples were homogenized in Nuclei EZ lysis buffer (NUC-101, Sigma-Aldrich) as recommended in presence of 0.25U/ul RNAse inhibitor. Lysis was promoted by gentle mixing of homogenate using bore tips, followed by incubation for 5 minutes on ice and passing of homogenate through 70um cell strainer. Nuclei were then pelleted by centrifugation of the filtered homogenate at 500g for 5 minutes at 4 degrees. Nuclei was washed two times with 500ul Nuclei Wash and Resuspension Buffer (1x PBS, 1% BSA, 0.25ul RNAse) for 5 minutes on ice (first wash without resuspending nuclei, second wash with resuspending nuclei). After each wash, nuclei were pelleted by centrifugation at 500g for 5 minutes at 4 degrees. Pelleted nuclei were further resuspended in 100-500ul Nuclei Wash and Resuspension Buffer with DAPI (10ug/ml) and nuclei were sorted to eliminate cellular debris. Sorted nuclei were then pelleted at 500g for 5 minutes at 4 degrees and 500ul ATAC wash buffer (10mM Tris-HCL, 10mM NaCl, 3mM MgCl2, 1% BSA, 0.1% Tween-20) was gently added and incubated for 5 minutes on ice without resuspending nuclei. Washed nuclei were then pelleted by centrifugation at 500g for 5 minutes at 4 degrees and nuclei was gently resuspended in 500ul ATAC wash buffer and incubated for 5 minutes. Nuclei pellet was obtained by centrifugation at 500g for 5 minutes at 4 degrees and resuspended in 100ul 1x TD buffer (20034198, Illumina). About 10K nuclei was used for transposition reaction. Nuclei were resuspended in 50ul transposition mixture, containing 2.5uL transposase in 1x TD buffer (20034198, Illumina) with 0.001% Digitonin and 0.01% Tween 20, and incubated for 30 minutes on a thermomixer at 1000RPM and 37C. DNA was extracted using MinElute Reaction Cleanup Kit (Qiagen).