GSM6690589: Control (Replicate 2); Mus musculus; RNA-Seq
Nuclei were acutely purified from brain cortex as described previously with minor modifications (Cid et al., 2021). Brains were immediately removed into ice cold HBBS 1x. A total of four libraries were generated; two libraries per condition (CrT+/y and CrT-/y) were prepared, corresponding to independent biological replicates. For each library, the cerebral cortex of the right hemisphere was manually dissected and transferred into 1 mL of ice-cold MACS buffer (0,5% BSA, 2 mM EDTA, PBS 1x). Each cortex was then homogeneized 12-15 times with the pestle of a dounce homogeneizer (20404, Lab Unlimited, Dublin, Ireland). Cell suspensions were passed through a 40 m cell strainer, collected in a 2 mL tube and centrifuged for 15 min (500G, 4ºC). The resulting cell pellets were resuspended in 1 mL of lysis buffer (10 mM Tris-HCL, 10 mM NaCl, 3 mM MgCl2, 0,1% IGEPAL) and transferred into a 15 mL falcon tube containing 9 mL of cell lysis buffer. The cell suspension was kept 5 min on ice, inverting the tube to mix the suspension every 1 minute. Samples were then spun down at 500G for 30 min in a pre-chilled centrifuge. The nuclei pellet was resuspended in PBS 1x 1% BSA, RNase inhibitor 0,2 U/L (3335399001, Merck, Darmstadt, Germany) and 15000 nuclei were purified by flow cytometry in a BD FACS Aria III (BD Biosciences, Madrid, Spain). The whole process was carried out at 4ºC. Purified intact nuclei were processed through all steps to generate stable cDNA libraries. For every sample, 15000 nuclei were loaded into a Chromium Single Cell A Chip (10x Genomics, Pleasanton, USA) and processed following the manufacturer's instructions. Single-nuclei RNA-seq libraries were prepared using the Chromium Single Cell 3' Library & Gel Bead kit v2 and i7 Multiplex kit (10x Genomics). Pooled libraries were then loaded on a HiSeq2500 instrument (Illumina) and sequenced to obtain 75 bp paired-end reads following manufacturer instructions. Libraries were sequenced to obtain more than 900 million reads in total (WT_1:>240M; WT_2:>238M; KO_1:>300M; KO_2:>209M). snRNA-seq