Data för: Persistent and Compartmentalized T cell Immunity in ALS

Deltagarna rekryterades genom ALS Clinical Research Centre på Karolinska Universitetssjukhuset i Stockholm, Sverige.

Human material: Immune cells isolated from cerebral spinal fluid (CSF) and blood. 17 paired CSF samples and blood samples were collected from patients with newly diagnosed ALS, 4 paired blood and CSF samples were collected from ALS follow ups. Singe cell sequencing platform: 5’ scRNA-seq + V(D)J TCR repertoire sequencing using the 10x Genomics platform. For more general inofrmation on the 10x Genomic platform please see here: https://www.10xgenomics.com/support/single-cell-immune-profiling User guids for 5’ scRNA-seq and V(D)J TCR repertoire can be found here respectively: https://assets.ctfassets.net/an68im79xiti/1ihALQkNrgD83PmBYZ4SrW/e94dbc40c2a6c64940ebc82eca9e9334/CG000507_ChromiumSingleCell5_v2_FeatureBarcode_Automation_UG_Rev_A.pdf https://assets.ctfassets.net/an68im79xiti/2JzjFJTfslSYe6GF9eh5TL/c228c4cc6b84c4f17fc6c25c8d7a5448/CG000207_ChromiumNextGEMSingleCellV_D_J_ReagentKits_v1.1_UG_Rev_G.pdf

Sample collection and processing Fresh CSF and blood samples were collected from ALS patients at the time of diagnosis for scRNAseq analysis. Blood samples (3.5 mL) were collected in EDTA vacutainers (BD), and CSF (20 mL) was collected via lumbar puncture using two 10 mL plastic tubes (Sarstedt). Samples were transported and processed within 2 hours of collection. CSF samples were centrifuged at 300×g for 10 mins at 4 °C. The supernatant was carefully removed without disturbing the cell pellet, which was resuspended in 50 µL PBS containing 0.04% bovine serum albumin (BSA). Peripheral blood mononuclear cells (PBMCs) were isolated using the prepTM (STEMCELL) protocol, following the manufacturer’s instructions. After centrifugation, PBMCs from the interface layer were collected and washed twice in 15 mL PBS supplemented with 0.04 % BSA. Cell counts were determined using a Fuchs-Rosenthal chamber (Biosigma) with 5 µL of single-cell suspension. CSF samples with visible blood contamination or >200 RBCs/µL were excluded. A total of 10,000 PBMCs and all available CSF cells per patient were used for library preparation. Library preparation and sequencing Single-cell suspensions were loaded onto Chromium Next GEM Chip K, and gel beads-in-emulsion (GEMs) were generated using the Chromium iX instrument (10x Genomics) according to the Chromium Next GEM Single Cell 5’ Reagent Kits v2 (Dual Index) protocol. cDNA amplification and library preparation were performed according to the manufacturer’s instructions, generating 5’ primer gene expression libraries and V(D)J libraries. Gene expression (GEX) libraries were pooled and sequenced at a target depth of ~ 50,000 reads per cell, while V(D)J libraries were sequenced at ~ 5,000 reads per cell using the Illumina NovaSeq6000 platform at Eukaryotic Single Cell Genomics (ESCG), SciLifeLab, Karolinska Institutet. The data set contains information from 21 individuals (17 ALS + 4 follow ups) using single cell RNA sequencing in combination with TCR V(D)J sequencing to study the immune profile of the central nervous compartment (CSF) and blood. Sequencing was done using 10x Genomics platform (5’ scRNAseq & V(D)J Reagent Kits v 2). 5’ and TCR libraries were then pooled and for sequencing on the NovaSeq sequencer. Provided files are in .fastq.gz format and per individual four files are available (Read 1&2 and Lane 1&2).