GSM4970087: VE66730: patient with viral encephalitis (VE), patient#VE66730; Homo sapiens; RNA-Seq
CSF samples were collected into round bottom polypropylene tubes and then centrifuged for 10 min at 300 x g. The CSF supernatant was removed and CSF cells were resuspended in 5 ml of X-Vivo15 media (Lonza) and stored at 4°C. The samples were then transported to the laboratory of the Department of Neurology with Institute of Translational Neurology at the University Clinic Münster, Germany for scRNA-seq. Transport time of samples collected in Münster was 10-30 minutes. Transport time of samples collected in Essen was < 60 minutes. Cell suspensions were then centrifuged again for 5 min at 400 x g and then resuspended in 40 μl of media. Out of this volume, 6 μl were used for cell counting and the remaining volume was entirely used for scRNA-seq. Four Neuro-COVID samples were processed with the Chromium Single Cell 5' Library & Gel Bead Kit and barcoded, full-length VDJ segments from T cells were enriched from first-strand cDNA via PCR amplification with the Chromium Single Cell VDJ Enrichment Kit, Human T Cell (from 10X Genomics) following the manufacturer's instructions. Further library preparation was performed using the Chromium Single Cell 5' Library Construction Kit (10X Genomics). Three Neuro-COVID samples were processed with the Chromium Single Cell 3' Library & Gel Bead Kit and we developed a novel method to sequence antigen receptor information  from 3' scRNA-seq libraries. The method allows removing the constant region of antigen receptors during enrichment, while maintaining their cell barcode and unique molecular identifier (UMI) information attached to the 3' of the cDNA molecules. In summary, the method involves self-circulating the cDNA library, enriching the VDJ region and re-linearizing. Each step maintains the VDJ region together with the cell barcode and UMI in the same molecule. Sample processing and library preparation was performed according to manufacturer instructions using AMPure XP beads (Beckman Coulter). Sequencing was either carried out on a local Illumina Nextseq 500 using the High-Out 75 cycle kit with a 26-8-0-57 read setup or commercially (Microanaly, China) on a NovaSeq 6000 using the 300 cycle kit with paired end 150 read setup. All the samples were sequenced with a sequencing depth >50,000 reads per cell.