title: GSM9042958: HT-29-et, pooled virus infection, 4dpi, biol rep2, targeted; Homo sapiens; RNA-Seq
library_construction_protocol: For single cell RNA sequencing, cells were harvested using TrypLE, resuspended in PBS, filtered through 30 µm  filters, facs sorted into PBS+1% FBS and directly processed into a library. Gene expression (GEX) libraries were created from 10000-30000 cells, which were sorted using FACSAria (BD Bioscience) and processed right after sorting following the standard Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (10X Genomics), following standard dual-index protocols and the Chromium-X controller (10xGenomics). Libraries were sequenced on the Nova Seq X Plus 25B with 150 PE (Illumina, Inc.) Barcode-targeted libraries were generated from amplified cDNA used for GEX libraries. PCR was performed with a primer (5′-GTGGTGTTAATTAACCTAGAGGGC-3′) carrying a specific handle and partial Read One primer to amplify the 10 bp viral barcode. Each 100 µL reaction included 100 ng cDNA, primers, and KAPA HiFi mix, with cycling: 95 °C (3 min), 5 cycles of 98 °C (20 s), 67 °C (60 s), 72 °C (60 s), and a final 72 °C (5 min). PCR products were size-selected with SPRIselect beads (0.6X/1.2X) per 10X Genomics protocol. For index PCR, 30 µL of cleaned product underwent 14 cycles, followed by standard indexing and clean-up. Final product size (~360 bp) was confirmed via TapeStation (Agilent). Sequcing was performed as for GEX libraries.