GSM9207650: SC; Mus musculus; RNA-Seq
CD45+ cells were isolated from the dLNs and spinal cords of EAE-induced C57BL/6 mice by fluorescence-activated cell sorting (FACS). Single-cell RNA-Seq libraries were prepared using SeekOne® Digital Droplet Single Cell 3' library preparation kit (SeekGene, Catalog No. K00202). Briefly, appropriate number of cells were mixed with reverse transcription reagent and then added to the sample well in SeekOne® chip S3. Subsequently Barcoded Hydrogel Beads (BHBs) and partitioning oil were dispensed into corresponding wells separately in chip S3. After emulsion droplet generation reverse transcription were performed at 42℃ for 90 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 amplify the DNA representing 3' polyA part of expressing genes which also contained Cell Barcode and Unique Molecular Index. The indexed sequencing libraries were cleanup with VAHTS DNA Clean Beads (Vazyme, N411-01), analyzed by Qubit (Thermo Fisher Scientific, Q33226) and Bio-Fragment Analyzer (Bioptic, Qsep400). The libraries were then sequenced on illumina NovaSeq 6000 with PE150 read length or DNBSEQ-T7 platform with PE150 read length.