Instrument: Illumina NovaSeq 6000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Cells cultured in Multidishes with UpCell™ Surface (Thermo Fisher) were detached by 15 min of incubation at 4°C to obtain single-cell suspensions. Approximately 2 million cells per experimental condition were then stained with 1 μg of TotalSeq™ B0301-B0308 anti-mouse Hashtag antibodies (BioLegend) according to the manufacturer's instructions and pooled together at equal cell numbers. The pooled multiplexed sample was then processed according to the 10x Genomics Chromium Single Cell 3' v3.1 Reagent Kits with Feature Barcoding Technology for Cell Surface Protein instruction guide. For all experiments, the sample volume was adjusted to a target capture of 10,000 cells and loaded on the 10x Genomics chromium next-GEM chip G to generate gel-beads-in-emulsion (GEMs). The GEM solution was placed in a thermal cycler for reverse transcription as described by the 10x Genomics instruction guide (53:00 min at 53°C followed by 5:00 min at 85°C). The resulting barcoded cDNA was then cleaned using Dynabeads MyOne Silane and amplified for 11 cycles (as recommended by the 10x Genomics user guide for a target cell recovery of >6,000 cells). After amplification, for multiplexed experiments, cDNA generated from polyadenylated mRNA for the 3' gene expression library was separated from DNA from the Cell Surface Protein Feature Barcode for the Cell Surface Protein library with Dynabeads MyOne Silane and SPRIselect reagents based on size. The quality and concentration of both cDNA and DNA were assessed using High-Sensitivity D5000 ScreenTape (Agilent). All samples presented product sizes with a narrow distribution centered around 2000 pb. cDNA and DNA were then subjected to enzymatic fragmentation, end repair and A-tailing. Adaptors were ligated to the fragmented cDNA and DNA, and the sample index was added during sample index PCR (set for 12 cycles, as recommended by the 10X Genomics user guide to correlate with a cDNA/DNA input of 12-150 ng). Library quality and concentration were assessed using High-Sensitivity D5000 ScreenTape (Agilent). All libraries showed an average fragment size of around 400 pb. For multiplexed runs, 3ʹ Gene Expression and Cell Surface Protein libraries were pooled at a ratio of 4:1 and sequenced using the Illumina NovaSeq 6000 system with a sequencing depth of 50,000 and 12,500 reads per cell, respectively, following the recommendations of 10X Genomics (paired-end reads, single indexing, read 1 = 28 cycles, i7 = 8 cycles, i5 = 0 cycles and read 2 = 91 cycles). For non multiplexed runs, 3ʹ Gene Expression libraries for each sample were pooled at an equimolar amount and sequenced using the Illumina NovaSeq 6000 system with a sequencing depth of 50,000 reads per cell, following the same recommendations of 10x Genomics.