GSM7059624: VDJ_IEL_Winkler_gd; Mus musculus; RNA-Seq
Mouse ears separated into dorsal and ventral halves and digested in RPMI medium with collagenase IV and DNase I at 37 °C. Cells were filtered and further enriched using a Percoll gradient. Cells from skin preparations were further barcoded with antibody hashing (Biolegend) for the respective genoytpes according to manufacturer's protocol (0,1µg/1*10^6 cells in 100 µl). In total , 19 mice were pooled,  7 of which were marked with Hashtag#1 as TCR𝛿-WT mice and 12 with Hashtag#2 as TCR𝛿-KI mice. γδ T cells were identified and FACS-sorted as DAPI-TCR𝛽-CD3+TCR𝛿+ and used for the 10X workflow. The pooled samples were then subjected to transcriptome and VDJ repertoire sequencing. 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 and further enriched using a Percoll gradient. Cells from IELpreparations were further barcoded with antibody hashing (Biolegend) for the respective genoytpes according to manufacturer's protocol (0,1µg/1*10^6 cells in 100 µl). In total , 19 mice were pooled,  7 of which were marked with Hashtag#1 as TCR𝛿-WT mice and 12 with Hashtag#2 as TCR𝛿-KI mice. γδIELs were identified and FACS-sorted as DAPI-TCR𝛽-CD3+TCR𝛿+ and 10,000 cells from TCR𝛿-WT TCR𝛿-KI each were used form the 10X workflow. The pooled samples were then subjected to transcriptome and VDJ repertoire sequencing. After sorting, viable cell numbers were determined and subjected to 10X library preparation (Next GEM Single Cell 5' Lib Kit V1.1) according to the manufacturer's protocol using the 10X Chromium device. After generation of full length 10X barcoded cDNA, a part of the cDNA library was subjected to enrichment amplification of V(D)J transcripts according to manufacturer's protocol with custom primers for TCRγ (round 1: TCRG_Cg1_2_Outer, TCRG_Cg3_Outer, TCRG_Cg4_Outer at 10mM concentration, round 2: TCRG_Cg1_3_Inner, TCRG_Cg4_Inner at 20mM concentration) and TCRδ (round 1: TRD_Outer at 10mM concentration, round 2: TCRD Inner at 20mM concentration) constant regions. The 5'GEX library was consequentially sequenced on a HiSeq platform with 100/8/100 bp and 28/9/91 bp parameters. The corresponding VDJ library was run twice on a HiSeq platform with 100/8/100 bp parameters.