GSM7665932: N2W 10x Multiome - ATAC; Mus musculus; ATAC-seq
Each biological replicate was minced separately and placed into a tube containing cold Nuclei PURE Lysis Buffer and 10% Triton X-100 (Sigma, NUC201-1KT). The minced tissue was transferred into a 7 ml ice-cold tissue grinder (Sigma, D9063), homogenized up and down 20-25 times, and filtered on ice through cell strainers of 100μm, 70μm and 40 μm (Pluriselect, 43-10040). RNasin Plus diluted 1/200 (Promega, N2611) was added in all solutions. Single -nuclei isolation for multiome sequencing experiments: After centrifuging at 500 rpm for 5 min at 4°C, the supernatant was aspirated and gently resuspended in 100 μl of a cold lysis solution containing Nuclei PURE Lysis Buffer, 10% Triton X-100, 0.01% of digitonin (ThermoFisher, BN2006), and 1% nuclease-free UltraPure™ BSA (ThermoFisher, AM2616). After 1 minute of incubation, 200 μl of cold 1X Nuclei Buffer (10x Genomics, 2000153/2000207) was added, the suspension was filtered again with a 40 μm cell strainer, centrifuged at 500 rpm for 5 min at 4°C, and gently resuspended into a final volume of 50 μl of cold 1X Nuclei Buffer. Single -nuclei isolation for RNA sequencing experiments: After centrifuging at 500 rpm for 5 min at 4°C, the supernatant was aspirated and gently resuspended in 400 μl of a cold wash solution containing 1X Hanks' Balanced Salt Solution HBSS and 1% nuclease-free UltraPure™ BSA. This step was repeated for a total of two times, and nuclei were resuspended into a final volume of 50 μl of cold wash solution. Single-nuclei libraries were generated using the Single Cell Multiome ATAC + Gene Expression kit (10x Genomics, PN-1000283). Manufacturer's instructions were followed for Tn5-based transposition, cell capture, barcoding, reverse transcription, cDNA amplification, and ATAC and RNA libraries construction. For wild-derived mice, 4 of 6 biological replicates for aPir, 1 of 4 for pPir, and 1 of 2 for SSp were processed using the Single Cell 3' Reagent Kits v3.1 dual index (10x Genomics, PN-1000268). Final libraries (40 libraries from gene expression and 32 libraries from ATAC experiments) were evaluated for quality and quantified using the Qubit fluorometer. The fragment size distribution was evaluated by Agilent TapeStation 2200 (Agilent Technologies). Libraries were further evaluated for proper incorporation of the Illumina adaptors on the Roche LightCycler 480 using the Roche Kapa library quant assay according to manufacturer's protocol.