title: GSM5379860: WPRTCR: wt-R-par TCR TCR-seq; Mus musculus; OTHER
library_construction_protocol: After fluorescent labeling of blood leukocytes, peak of disease AT-EAE recipient mice (of either wt-Th17 or Bcl6KO-Th17 donor cells) were anesthetized and intracardially perfused, and spinal cord (SC) meninges and parenchyma were extracted separately. Parenchymal tissue was flushed out from the spinal canal with hydrostatic pressure. SC meninges remained attached to the vertebrae and were peeled of under the binocular. Parenchymal tissue was digested with collagenase D/DNase I and cells were isolated via 70%/37% percoll gradient as described before. Meningeal cell were extracted using collagenase D for digestion. Isolated cells were sorted either for CD45highCD45iv- cells or Vβ11+CD4+CD45highCD45iv- cells. In vitro differentiated Th17 cultures were sorted for Vβ11+CD4+ cells. Single cell suspensions of CD45highCD45iv- cells (5 μl each) were manually counted in a Fuchs-Rosenthal chamber and the cell concentration was adjusted to achieve a cell recovery of approx. 5'000-6'000 cells. In total, 5 wt-R AT-EAE mice vs. 5 Bcl6KO-R AT-EAE mice were used to generate single cell RNA sequencing data from SC meningeal vs. parenchymal tissues. Per tissue, an average of 4,017 ± 132 SEM cells were used for analysis. 57,846 ± 1,060 SEM reads per cell were sequenced and this resulted in 2136 ± 176 SEM median genes detected per cell. In addition, we did bulk RNA sequencing on sorted Vβ11+CD4+ of SC meningeal (5 wt-R AT-EAE mice vs. 5 Bcl6KO-R AT-EAE mice) or parenchymal (4 wt-R AT-EAE mice vs. 4 Bcl6KO-R AT-EAE mice) CD45highCD45iv- cells or sorted Vβ11+CD4+ of in vitro differentiated Th17 cells (5 wt-Th17 vs. 5 Bcl6KO-Th17 in vitro cultures). On average, 15,19 ± 0,65 SEM M reads were sequenced and used for bulk-seq analysis. 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) with v3 chemistry. For TCR 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 Novaseq 150-8-8-150 read setup (scRNA-seq and TCR-seq) and on a NextSeq500 using the High-Out 75 cycle kit (Illumina) with 0-8-8-75 set up (bulk-RNA seq).
attributes.tag: GEO Accession
attributes.value: GSM5379860