alias: GSM3679891
title: GSM3679891: pooled_Vγ6; Mus musculus; RNA-Seq
library_construction_protocol: After euthanasia, ears, thymi and peripheral lymph nodes (pLN; including superficial cervical, axillary, brachial and inguinal) were collected.    To isolate lymphocytes from ear skin, ears were split into dorsal and ventral layers, cut into small pieces, and digested in RPMI media supplemented with (2 mg/ml) collagenase IV (Worthington) and (187.5 µg/ml) DNaseI (Roche) for 75 minutes at 37°C under 1400 rpm of shaking. The digestion was terminated by 5mM EDTA. Digested ears repeatedly went through an 18G needle for further dissociating tissue structure and releasing cells. Subsequently cells were filtered (100 μM cell strainer (Falcon) and facilitated to density gradient centrifugation using 40% and 70% Percoll solutions. For downstream applications cells were re-suspended in MACS buffer (PBS with 3% FCS and 4mM EDTA).  Single cell suspensions from thymus and peripheral lymph nodes were obtained by filtering organs through nylon gaze. To enrich thymus samples γδ T cells, CD4+ and CD8+ T cells were depleted. For flow cytometric cell sorting of Vγ6+ T cells and subsequent single-cell RNA-seq library generation, CD4+ and/or CD8+ thymus cells were lysed via incubation with monoclonal anti-CD4 and anti-CD8β IgM (homemade), DNaseI (Roche) and Standard Rabbit Complement (Cedaralane) at 37°C for 30 minutes. Next, thymus cells were retrieved via density gradient centrifugation using Lympholyte M (Cederlane). FACS-Aria II (BD Bioscience) was used for cell sorting of indicated cell populations into PBS/4mM EDTA/5%FCS. Sorted Vγ4+  and Vγ6+  γδTcells were diluted to ≤ 1000 cells/μL. Single cell sequencing libraries were generated using the Chromium™ Single Cell 3' reagent kits v2 according to the manufactures protocol (10xGenomics). Indexed libraries were pooled, and paired-end sequencing was performed by Hiseq4000 (Illumina).
identifiers.namespace: GEO
identifiers.id: GSM3679891