GSM4261910: Hippocampal_area_CA1_epilepsy; Mus musculus; RNA-Seq
Single nuclei from 2 control and 2 epileptic Calb1::CrexTdtomato young adult male mice were acutely isolated. Animals were sacrificed 12 weeks after the kainate/saline administration by cervical dislocation and brains were dissected and cut in 300um thick slices in a vibratome covered by ice cold HBBS1x. Dorsal CA1 region were manually dissected from 4 consecutive slices and put altogether into 400uL of ice cold MACS buffer (0,5%BSA, 2mM EDTA, PBS1x). CA1 portions were transferred to a dounce homogenizer (20404 Lab Unlimited) containing 400uL of MACS buffer and were homogenized 12-15 times each with the pestle. The cell suspension was transferred to a 2mL Eppendorf tube and centrifuge 15min 500G 4ºC. Cell pellets were resuspended in 500uL of lysis buffer (10mM Tris-HCL, 10mM NaCl, 3mM MgCl2, 0,1% IGEPAL) and kept 5 minutes on ice. Samples were then spun down at 500xg for 30 min in a pre-chilled centrifuge. The nuclei pellet was resuspended in PBS1x 1%BSA and then 15,000 nuclei were sorted on a BD FACS Aria III. The whole process was carried out at 4ºC. FACS purified nuclei from mouse hippocampal CA1 area were processed through all steps to generate stable cDNA libraries. For every sample, 15,000 nuclei were loaded into a Chromium Single Cell A Chip (10x Genomics) 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.
GEO Accession
GSM4261910