GSM7037563: TRD_TRG, spleen, WT, replicate 3; Mus musculus; RNA-Seq
To retrieve single cell suspensions from spleen, freshly-isolated organs were injected with Collagenase D/DNAse I solution (2mg/ml and 1mg/ml) with a syringe followed by a 15-minute incubation at 37°C. The organs were transferred into GentleMACS C-Tube containing 3ml PBS and subjected to the pre-defined “37 SDK” program in the gentleMACS Octo Dissociator. Retrieved cell suspensions were filtered through a 70 𝜇m filter and cells were pelleted, washed with PBS and subjected to erythrocyte lysis. Small intestines were removed and cleaned of connective tissue, Peyer's patches and luminal content. Intraepithelial cells were isolated by incubating in an EDTA-containing buffer at 37 °C and subsequent vigorous shaking. γδ T cells from spleen and the small intestinal epithelium were identified as CD3+ γδTCR+ αβTCR- and subjected to flow cytometry sorting (Astrios II). Cells were directly sorted into lysis buffer and stored at -20 °C until RNA extraction and reverse transcription to cDNA. To apply a molecular amplification fingerprinting (MAF) strategy individual transcripts were labelled during cDNA synthesis. Thus, primers binding to the constant segment of TRG and TRD chains were used which included a Reverse Identifier (RID) and Illumina Adapter sequences for cDNA synthesis. TRG and TRD reads were transcribed from RNA to cDNA. cDNA products were cleaned up in three consecutive rounds with SPRI beads (Ampure/Agencourt) and subjected to multiplex PCR. V-gene specific primers included Forward Identifiers (FID) for correcting amplification and sequencing bias as well as Illumina Adapter sequences. The PCR products from the multiplex DNA step were cleaned up with SPRI beads (Ampure/Agencourt). After clean-up of the PCR products, individual sample indices were added to the PCR amplicons using Illumina Nextera i5 and i7 primers. The cleaned cDNA was then used as template for sample indexing via adding unique sample indices (Illumina Nextera Kit). At each step, success and quality of the PCRs were verified using gel electrophoresis