GSM3898394: dKAT3_30d_snRNAseq_1; Mus musculus; RNA-Seq
Hippocampi were micro-dissected after cervical dislocation, washed in ice-cold PBS and pooled together, when fixation was needed 1% FA was used. Nuclei were isolated using our improved FANS protocol for SUN1-GFP+ cells. Extensive description of protocols indicated in Supplemental Experimental Procedures. For snRNAseq, the isolation of single nuclei was carried out at 4ºC. The two hippocampi of each mouse were dissected in cold PBS and transferred to a dounce homogeneizer containing 1 mL of ice cold MACS buffer (0.5% BSA, 2 mM EDTA, PBS 1x) and were homogenized 15-12 times with the pestle. The cell suspension was transferred to a 2 mL tube and centrifuge 15 min at 500g and 4ºC. Cell pellets were resuspended in 2 mL of lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 0,1% IGEPAL) and kept 5 min on ice. 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 and sorted on a BD FACS Aria III. 15,000 nuclei per sample were loaded into the single cell A Chip and then generation of barcode-containing partitions were carried out with the Chromium Controller (10X Genomics). snRNAseq libraries were prepared according to manufacturer instructions using Chromium Controller. Chromium Single Cell 3' Library & Gel Bead Kit v2 and Chromium Single Cell A Chip Kit (10x Genomics) snRNAseq library construction protocol: Chromium Single Cell 3' Library & Gel Bead Kit v2 was employed for post-GEM-RT cleanup, cDNA amplification and the generation of barcoded (ChromiumTM i7 Multiplex Kit) libraries. Libraries were sequenced to an average depth of 290-310 Million reads per sample on an Illumina HiSeq2500 sequencer according to manufacturer instructions. Quality control of sequenced reads was performed using FastQC (Babraham Institute). Sequenced samples were processed using the Cell Ranger v2.2.0 pipeline (10x Genomics) and aligned to the CRGm38 (mm10) mouse reference genome customized to count reads in introns (pre-mRNA) (gene annotation version 94). We retrieved between 1.791 (control), 1.133 (dKAT3-ifKO 2w), and 1.465 (dKAT3-ifKO 1m) high quality nuclei per sample. Mean reads per nuclei were 172.879 (control), 271.535 (dKAT3-ifKO 2w), and 197.840 (dKAT3-ifKO 1m). snRNAseq barcodes correspond to nucleotides 59-74 of read 1 (R1 files)
GEO
GSM3898394