GSM7968348: PBMC_Patient_2_Pre; Homo sapiens; RNA-Seq
Prior to single cell RNA sequencing, patient PBMCs were processed and frozen by the NIH biobanking resource as described in Heinrich et al. 2022. PBMC samples were thawed at 37°C for rapid recovery and washed with FACS buffer (BD, Cat. No. 554656). Cell count and viability were assessed on a CellometerAuto200 (Nexcelom Bioscience). Prior to single cell sequencing, live cells were purified by magnetically depleting dead cells using a Dead Cell Removal Kit  (Miltenyi Biotec, Cat.No. 130-090-101) and sorted using an AutoMACS Pro (Miltenyi Bitoec) following the manufacturer's instructions. Next, sorted cells were instantly transferred for single-cell cell capture and library preparation. For bulk RNA sequencing, RNA was extracted from fresh-frozen needle biopsy tumor samples at baseline and post-treatment, whereas RNA integrity and quantitation were assessed using the RNA Nano 6000 Assay Kit of Bioanalyzer 2100 system (Agilent Technologies, California, USA).  Heinrich, B., et al. The tumour microenvironment shapes innate lymphoid cells in patients with hepatocellular carcinoma. Gut 71, 1161-1175 (2022). 10x Genomics Single Cell Chromium 5' mRNA libraries were prepared as instructed by the 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). The scRNA-Seq samples were counted and diluted in PBS + 0.04% BSA, whereas the sample concentration and viability were assessed using the LunaFL fluorescent cell counter. The cellular samples were loaded in the lanes on the Chromium Connect according to the 10X Genomics 5' v2 Single Cell User Guide with a single capture lane per sample targeting recovery of up to 6,000 cells per lane. Cell partitioning completed successfully with uniform emulsion consistency. All steps of the library preparation were performed on the Connect and quality control were performed as described in the 10X Genomics 5' v2 Single Cell User Guide. Bulk RNA-Seq libraries were prepared using the NEB next Ultra RNA Library Prep Kit for Illumina (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using PE Cluster Kit cBot-HS (Illumina) according to the manufacturer's instruction.