GSM8835514: TCRTg_HTO; Mus musculus; OTHER
For preparation of transgenic cells for single-cell multiome sequencing, congenically marked naïve TCRTg cells (M45Tg and M38Tg) were transferred into congenically distinct naïve recipient mice prior to infection with MCMV (see Methods), as illustrated in Extended Data Fig. 4A. On the day of cell isolation, each mouse was injected with 50µg of anti-ARTC2 nanobody (i.p.) 30 minutes before sacrifice (BioLegend, Catalog No. 149803, Clone S+16a) and processed as described above (see Tissue processing and preparation of single cell suspension). Intravenous (I.V.) labeling was not performed prior to sorting due to its limited ability to distinguish circulating from bona fide tissue-resident cells in the liver. Notably, I.V. labeling showed minimal signal in the salivary gland, comparable to the lymph nodes, which remain unlabeled (data not shown). Upon cell sorting into 1× PBS + 0.04% BSA, TCRTg cells were stained with mouse TotalSeq-A Hash Tag Oligonucleotides antibodies (BioLegend, tag number A0301-A0310), oligo-tagged anti-rat/mouse CD90.1 (BioLegend, Catalog No. 202547, Clone OX-7), and anti-mouse CD90.2 (BioLegend, Catalog No. 105345, Clone 30-H12). Hash tagged cells were then pooled at approximately equal ratios for multiome sequencing. Single Cell Multiome ATAC + Gene Expression was performed with 10X Genomics system using Chromium Next GEM Single Cell Multiome Reagent Kit A (Catalog No. 1000282) and ATAC Kit A (Catalog No. 1000280). We followed Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Kits User Guide with the difference that single cell suspension was prepared as described(Mimitou et al., 2021) (DOGMA-seq protocol with Digitonin buffer). Briefly, cells were treated with a digitonin lysis buffer (20mM Tris-HCl pH 7.4, 150mM NaCl, 3mM MgCl2, 0.01% DIG and 2U/µl of RNase inhibitor) for 5 mins on ice, followed by adding 1ml of chilled DIG wash buffer (20mM Tris-HCl pH 7.4, 150mM NaCl, 3mM MgCl2 and 1U/µl of RNAse inhibitor) and inversion before centrifugation at 500g for 5 mins at 4 °C. The supernatant was discarded, and cells were resuspended in DIG wash buffer, followed by counting using trypan blue and DAPI on a Countess II FL Automated Cell Counter. Cells were processed according to the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression user guide. In addition, samples were multiplexed together on one lane of 10X Chromium (using Hash Tag Oligonucleotides - HTO). Final libraries were sequenced on Illumina NovaSeq S4 platform (R1 – 28 cycles, i7 – 8 cycles, R2 – 90 cycles).