title: GSM5027155: single-cell RNA seq data of the choroid plexus from rat [CP]; Rattus norvegicus; RNA-Seq
library_construction_protocol: Cells from CNS, SDM, dura, and CP were extracted as described before and sorted for  CD45+CD45iv- cells. CSF cells were not sorted. Blood cells were not sorted, but purified by  gradient centrifugation on Lymphoprep (Stemcell) in accordance with manufacturer's  instructions. After centrifugation, peripheral blood mononuclear cells enriched in the interface  were taken off and washed in 10 ml of PBS with 2% FCS media. Single cell suspensions  from all tissues (5 μl each) were manually counted in a Fuchs-Rosenthal chamber and the  cell concentration was adjusted to achieve a cell recovery of approx. 6'000-10'000 cells.  In total, 25 naive Lewis rats were used to generate single cell RNA sequencing data from rat  tissues. Per tissue, an average of 4,384 ± 736 SEM cells were used for analysis. 51,278 ±  2,859 SEM reads per cell were sequenced and this resulted in 733 ± 100 SEM median  genes detected per cell. In addition, we did single cell RNA sequencing on the cells isolated  from the skull dura of 3 pooled C57BL/6 mice at the peak of active EAE and 4 pooled  unimmunized C57BL/6 mice as ctrl. On average, 3,900 ± 1,100 SEM cells per sample were  used for analysis. 60,541 ± 4,644 SEM reads per cell were sequenced and resulted in 1,736  ± 852 SEM median genes detected per cell. Single cell suspensions were loaded onto the Chromium Single Cell Controller using the Chromium Single Cell 3' Library & Gel Bead Kit (both from 10X Genomics). Rat experiments were done with v2 chemistry. The mouse experiments were done with v3 chemistry because of the manufacturer's reagent update. For BCR sequencing, we developed a method to sequence antigen receptor information from the 3' scRNA-seq libraries by 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. Sample processing and library preparation was performed according to manufacturer's instructions using AMPure beads (Beckman Coulter). Sequencing was carried out on a local Illumina Nextseq 500 using the High-Out 75 cycle kit with a 26-8-0-57 read setup and Novaseq 150-8-0-150 read setup (NextSeq: read1-26bp, read2-57bp. Novaseq: PE150bp).