GSM9126539: Hpdlf/-_Ctrl_biorep1; Mus musculus; RNA-Seq
Mice at postnatal day 12 were anesthetized by avertin and perfused with cold PBS. A hemisphere of the brain was rapidly dissected, frozen in liquid nitrogen and stored at −80°C until use. For nuclei preparation, all steps were performed on ice. Frozen brain tissue was minced in 1 mL nuclear lysis buffer (NST: 0.1% NP-40, 10 mM Tris-HCl pH 7.4, 146 mM NaCl, 1 mM CaCl₂, 21 mM MgCl₂, 1 U/μL RNase inhibitor), and lysed for 7 minutes. Complete lysis was confirmed by trypan blue staining. Lysis was stopped by adding 1 mL ST Wash buffer (same as NST without detergent, supplemented with 0.01% BSA and 40 U/mL RNase inhibitor). The lysate was filtered through a 40-µm cell strainer, washed with additional buffer, and centrifuged at 500 × g for 5 min at 4°C. The nuclear pellet was resuspended in PBS with 1% BSA, centrifuged again, and finally resuspended in 100 µL PBS/BSA. Nuclear integrity and concentration were assessed by trypan blue exclusion and microscopy. The nuclear suspension was diluted to a concentration of 700–1,200 nuclei/μL using PBS with 1% BSA. Single-nucleus RNA sequencing libraries were prepared according to the manufacturer's protocol for the 10× Genomics Chromium Next GEM Single Cell 3ʹ Reagent Kit v3.1 (Cat.no. PN-1000268). The constructed libraries were sequenced on the Illumina novaseq Xplus PE150 platform for high-throughput sequencing.