title: GSM6219844: Tumor1, CD8aapos, WT replicate 1; Mus musculus; RNA-Seq
library_construction_protocol: Single-cell RNA sequencing.  Cryopreserved single cell suspensions of human colon biopsies were thawed at 37 C, washed with RPMI, and rested in complete RPMI in 96-well plate for 1 hour. The cells were subsequently incubated with PMA/ionomycin for 3 hours, washed, blocked in Fc receptor block (TruStain FcX, Biolegend), and tagged with cell hashing antibody. Conjugation of purified antibodies for cell hashing was performed as described by the Technology Innovation Lab at the New York Genome Center (https://cite-seq.com/protocol/). The list of antibodies, clones, and barcodes are available in Table S3. The hashtagged samples were then pooled and stained for FACS sorting with anti-CD3 (clone OKT3, BioLegend) APC, anti-TCRγδ (clone 5A6.E9, Thermo Fisher) PE, and anti-TCRαβ (clone IP26, BioLegend) Alexa Fluor 488. Samples were subsequently stained with Sytox Blue (Thermo Fisher) prior to acquisition to determine cell viability. Viable T cells were sorted in FACS Aria II by gating SytoxBlue- CD3+TCRγδ+TCRαβ- cells and subsequently transferred into RPMI with 10% FBS at 4C. FACS-sorted CD3+ TCRγδ+ cells were loaded onto the Chromium 10x Genomics v1.1 platform (10X Genomics) as described in the manufacturer's protocol. Single cell 5' transcript and TCRγδ libraries were prepared using Chromium Next GEM Single Cell V(D)J Reagent Kits v1.1 with Feature Barcode Technology for Cell Surface Protein (CG000228). Following cDNA amplification, cell hashing additive oligos were added to amplify cell hashtag oligos. The products were separated using SPRIselect Beads (Beckman Coulter). Cell hashtag library generation was performed as described by the Technology Innovation Lab at the New York Genome Center (https://cite-seq.com/protocol/). TCRγδ libraries were prepared as described in Mimitou et al., 2019. cDNA libraries were quantified using Agilent High Sensitivity D5000 ScreenTape (Agilent, no. 5067-5592). Libraries were sequenced using NovaSeq 6000 platform. Bulk mRNA-Seq Library Preparation. Sorted cells (300-800 cells) were lysed in a guanidine thiocyanate buffer (TCL buffer, QIAGEN) supplemented with 1% β-mercaptoethanol. RNA was isolated by solid-phase reversible immobilization bead cleanup using RNAClean XP beads (Agentcourt, A63987), reverse transcribed, and amplified as described (57). Uniquely barcoded libraries were prepared using Nextera XT kit (Illumina) following manufacturer's instructions. Sequencing was performed on an Illumina NextSeq500.