Single-cell RNAseq of α-PD-1 relapsing YUMM2.1 tumours subsequently treated with PARP14i in vivo
Single-cell RNAseq of α-PD-1 relapsing YUMM2.1 tumours subsequently treated with PARP14i in vivo
Tumours were dissociated to achieve a single cell solution. Immune population was enriched by CD45 mouse Microbeads, and ~5% CD45 negative cells were spiked back into the CD45 positive cells. Cells were fixed for 18 hours, then quenched and stored in -80°C in Enhancer until all 16 samples were collected and sequenced using 10x Single cell flex. YUMM2.1 cells were subcutaneously injected into the left flank of mice. When tumours reached a volume of 80 mm3, mice were administered with two doses of α-PD-1 antibody i.p. Tumours were monitored until they reached 140 mm3 at which point a second round of treatments commenced: 2 doses of rat IgG2b (i.p.), or 2 doses of α-PD-1 antibody, or 14 doses of PARP14 inhibitor by oral gavage twice a day, or a combination of α-PD-1 i.p. and PARP14 inhubitor gavage in the same doses. Mice were culled on day 7 of re-treatment. Gene expression libraries were prepared from formaldehyde fixed samples using the Chromium X and Chromium Fixed RNA kit, Mouse Transcriptome, 4 rxns x 16 BC (10x Genomics, Inc. Pleasanton, USA) according to the manufacturer's protocol (CG000527 Rev C). Briefly, samples were incubated overnight with probe pairs designed to hybridise to mRNA, samples were pooled and washed to remove unbound probes. Nanoliter-scale Gel Beads-in-emulsion (GEMs) were generated by loading barcoded Gel Beads, a master mix containing pooled, probe-hybridised cells, and partitioning oil onto a Chromium Next GEM chip. Cells were delivered at a limiting dilution, such that the majority (90-99%) of generated GEMs contain no cell, while the remainder largely contain a single cell. The Gel Beads were then dissolved, primers, including an Illumina TruSeq Read 1 sequence, a 16-nucleotide 10x Barcode, a 12-nucleotide unique molecular identifier (UMI) and partial Capture Sequence 1, were released, and any co-partitioned cells lysed.  Illumina-compatible sequencing libraries were constructed by adding P5, P7, i5 and i7 sample indexes, and Illumina Small Read 2 sequences to the 10x barcoded, ligated probe products via Sample Index PCR followed by a SPRIselect size-selection.