title: GSM9266711: Testis,WT-biol rep1; Mus musculus; RNA-Seq
library_construction_protocol: Single-Nuclei Isolation Nuclei were isolated with Nuclei EZ Lysis buffer (NUC-101; Sigma-Aldrich) supplemented with protease inhibitor(5892791001; Roche) and RNase inhibitor(N2615; Promega and AM2696; Life Technologies). 1. Samples were cut into ~1 mm pieces and homogenized using a Dounce homogenizer (885302-0002; Kimble Chase) in 2 ml of ice-cold Nuclei EZ Lysis buffer. 2. They were incubated on ice for 5 minutes with an additional 2 ml of lysis buffer. 3. Grounded with a dounce(Sigma), resuspended by pipette，gentlely； 4. Incubated on ice for 6 min, then add 2 ml of ice-cold 4% BSA, resuspended by Pasteur pipette, then stop the reaction; 5. Centrifuged at 300g for 5 minutes at 4°C; 6. Add 2 ml of lysis buffer and 4% BSA, resuspended, Incubated on ice for 3 min; 7. De-fragment with Miltenyi (Debris Removal Solution); 8. The pellet was resuspended and washed with 4 ml of the buffer, and then, it was incubated on ice for 5 minutes. After another centrifugation; 9. The pellet was resuspended in Nuclei Suspension Buffer (1x PBS,0.07% BSA, and 0.1% RNase inhibitor); 10. Filtered through a 20-mm cell strainer(43-50020-50; pluriSelect), and counted using a haemocytometer/ Countess II Automated Cell Counter and concentration adjusted to 700-1200 cells/μl. Chromium 10x Genomics library and sequencing Single-nuclei suspensions were loaded to 10x Chromium to capture 8000 single cell according to the manufacturer's instructions of 10X Genomics Chromium Single-Cell 3' kit (V3) . The following cDNA amplification and library construction steps were performed according to the standard protocol. Libraries were sequenced on an Illumina NovaSeq 6000 sequencing system (paired-end multiplexing run,150bp) by LC-Bio Technology co.ltd., (HangZhou,China) at a minimum depth of 20,000 reads per cell.
identifiers.namespace: GEO
identifiers.id: GSM9266711_r1