GSM7109167: Patient1, PBMC, 12months; Homo sapiens; RNA-Seq
After harvested, tissues were washed in ice-cold RPMI1640 and dissociated using Multi-tissue dissociation kit 2from Miltenyi Biotec as instructions. DNase 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 (Miltenyi 130-094-183) 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 albuminat. Single-cell RNA-seq libraries were prepared using SeekOne® DD Single Cell 5' library preparation kit (SeekGene Catalog No.). Briefly, appropriate number of cells were mixed with reverse transcription reagent and then loaded to the sample well in SeekOne® DD Chip S5. Subsequently Gel Beads and Partitioning Oil were dispensed into corresponding wells separately in chip. After emulsion droplet generation reverse transcription were performed at 53°Cfor 45 minutes and inactivated at 85°C for 5 minutes. Next, cDNA was purified from broken droplet and amplified in PCR reaction. The amplified cDNA product was 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.