GSM7849813: scATAC-seq from Naïve Lungs; Mus musculus; ATAC-seq
For nuclei isolation, sorted cells were centrifuged at 300 rcf for 5 minutes at 4°C and then resuspended in 150uL of PBS supplemented with 0.04% BSA. A 100µL aliquot of this cell suspension was transferred to a 0.2mL flat-cap tube, centrifuged again under the same conditions, and subsequently resuspended in 50uL of Lysis Buffer (containing 10mM Tris-HCL, 10mM NaCl, 3mM MgCl2, 0.1% Tween 20, 0.1% Nonidet P40 Substitute, 0.01% Digitonin, and 1% BSA). This was incubated on ice for 4 minutes. Post-incubation, 50µL of Wash Buffer (comprising 10mM Tris-HCL, 10mM NaCl, 3mM MgCl2, 1% BSA, and 0.1% Tween 20) was added to the lysed cells. The nuclei suspension was then centrifuged at 500 rcf for 5 minutes at 4°C. The nuclei were washed with 45µL of a 1:20 dilution of the Nuclei Buffer (10x Genomics, PN-2000153/2000207) and centrifuged again using the same conditions. Finally, the isolated nuclei were resuspended in a volume of Diluted Nuclei Buffer to obtain a concentration ranging from 3080 to 7700 nuclei/µL. This was used as input for the 10X protocol (CG 000209 Rev D) targeting a recovery of 10,000 nuclei. The transposition reaction and library construction were performed following the protocol from 10X (CG000209 REV D). Sequencing was conducted on a NextSeq 500 with the parameters: Read 1N: 50 cycles, i7 index: 8 cycles, i5 index: 16 cycles, and Read 2N: 50 cycles.