GSM8666769: Brain, snRNAseq; Mus musculus; RNA-Seq
Intact nuclei were extracted using a protocol optimized for single-nucleus RNA sequencing (snRNA-seq) using the EasySci protocol adapted for mouse cortex tissue. The tissue was kept on dry ice, minced into fine pieces, and immediately placed in pre-chilled PBS (AM9624, Thermo Fisher Scientific) treated with 1% diethyl pyrocarbonate (DEPC, D5758-25ML, Sigma Aldrich). After several washes with ice-cold EZ Lysis Buffer (78425, Thermo Fisher Scientific) supplemented with RNase inhibitors (SUPERase In RNase Inhibitor, AM2694, Invitrogen), the tissue was mechanically disrupted using a syringe plunger and filtered through 40 µm cell strainers (CS4040, dot scientific inc.) to isolate nuclei. Nuclei were then resuspended in EZ Lysis Buffer supplemented with RNase inhibitors to ensure RNA integrity during processing. Formaldehyde was added at a final concentration of 0.1% (16% Formaldehyde Solution, 28906, Thermo Scientific) for 10 minutes to crosslink the nuclei. Following crosslinking, the nuclei were pelleted, washed, and stored at -80°C in a cryoprotective medium containing 10% dimethyl sulfoxide (DMSO, D8418, Sigma-Aldrich) for long-term storage until further processing. For library preparation, we followed the EasySci protocol. Nuclei were thawed and washed in Nuclei Storage Buffer (NSB) (Nuclease-free water, 10977-015, Invitrogen) supplemented with 0.1% Triton X-100 (93443-100ML, Sigma-Aldrich). The nuclei were counted and distributed into multi-well plates at a density of 20,000 nuclei per well. Reverse transcription was initiated using short-dT and randomN primers followed by the addition of reverse transcription reaction mix (Maxima Reverse Transcriptase, EP0741, Thermo Fisher Scientific). The reaction was carried out using a thermocycler with a multi-step temperature protocol for optimal reverse transcription. Afterwards, tagmentation was performed using a 1:100 dilution of Tagmentase and 2X Tagmentation Buffer (NEBNext Ultra II Q5 Master Mix, E7649AA, New England BioLabs). Following the addition of indexed P7 primers (E7335, New England BioLabs) and NEBNext Ultra II Q5 Master Mix (E7649AA, New England BioLabs), the samples were amplified through PCR to generate the final cDNA libraries. Libraries were purified using AMPure XP beads (A63882, Beckman Coulter) and checked for quality before sequencing. The libraries were quantified by qPCR using NEBNext Library Quant Kit for Illumina (E7642S, New England BioLabs) according to the manufacturer's instructions.