title: GSM8010571: Methamphetamine, replicate1, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: After harvested from mice,hippocampal tissues were washed in ice-cold RPMI1640 and dissociated using Tissue Dissociation Reagent A (Seekone K01301-30) from SeekGene as instructions. DNase Ⅰ (Sigma 9003-98-9) treatment was optional according to the viscosity of the homogenate. Cell count and viability was estimated using fluorescence Cell Analyzer (Countstar® Rigel S2) with AO/PI reagent after removal erythrocytes (Solarbio R1010) and then debris and dead cells removal was decided to be performed or not (Miltenyi 130-109-398/130-090-101). Finally fresh cells were washed twice in the RPMI1640 and then resuspended at 1×106 cells per ml in 1×PBS and 0.04% bovine serum albumin. Single-cell RNA-Seq libraries were prepared using Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (10x Genomics Catalog No.1000268). Briefly, appropriate number of cells were mixed with reverse transcription reagent and then loaded to the sample well in Chromium Next GEM Chip G. Subsequently Gel Beads and Partitioning Oil were dispensed into corresponding wells separately in chip. After emulsion droplet generation reverse transcription were performed at 53℃for 45 minutes and inactivated at 85℃ for 5 minutes. Next, cDNA was purified from broken droplet and amplified in PCR reaction. The amplified cDNA product was then cleaned, fragmented, end repaired, A-tailed and ligated to sequencing adaptor. Finally, the indexed PCR were performed to amplified the DNA representing 3' polyA part of expressing genes which also contained Cell Bar code and Unique Molecular Index. The indexed sequencing libraries were cleanup with SPRI beads, quantified by quantitative PCR (KAPA Biosystems KK4824) and then sequenced on illumina NovaSeq 6000 with PE150 read length or DNBSEQ-T7 platform with PE150 read length.