GSM7431767: Control mouse 2, snRNAseq; Mus musculus; RNA-Seq
Frozen PFC tissue (100 mg) was dounced in 1 mL of pre-cooled Nuclear Extraction Buffer (add RNase inhibitor and DTT before use, the final concentrations of the two are 0.2 U/µL, 1 mM, respectively) and left to lyse for 10 min, after which 9 ml of pre-cooled wash buffer was added and swirled. The sample was passed through a 40μm filter and spun for 5 min at 500 × g 4°C. This step was repeated two more times. After pelleting, the nuclei were gently resuspended in 5 – 10 mL of wash buffer by pipetting up and down 8 – 10 times. After 3 washes, the nuclei were resuspended in 200 µL of pre-cooled PBS (the PBS is added with RNase inhibitor and DTT before use, the final concentration of the two is 0.2 U/µL, 1 mM). Single nucleus suspensions (1×105 cells/mL) with PBS (HyClone) were loaded into microfluidic devices using the Singleron Matrix® Single Cell Processing System (Singleron). Subsequently, the snRNA-seq libraries were constructed according to the protocol of the GEXSCOPE® Single Nucleus Cell RNA Library Kits (Singleron). Individual libraries were diluted to 4 nM and pooled for sequencing on Illumina novaseq6000 with 150 bp paired end reads.