GSM6216318: scRNAseq_circFAT3_90d_1; Homo sapiens; RNA-Seq
After methanol fixation, cells were pelleted at 3000xg for 5 min and rehydrated in 1 ml PBS with 0.01% BSA and 1:100 Superasein. The cells were then resuspended in 300 μl rehydration buffer, containing PBS-BSA 0.1% plus 1 U/μl RNase Inhibitor (RiboLock; Thermo Fisher). Cells were filtered through a 40 μm cell strainer, counted using an automated cell counter (Countess; Thermo Fisher), and diluted to approximately 350 cells/μl in 250 μl rehydration buffer. Barcoded beads (ChemGenes, Macosko- 2011- 10 (V+)) with chilled lysis buffer (6% Ficoll PM-400, 0.2% Sarkosyl, 20 mM EDTA, 200 mM Tris pH 7.5, 50 mM DTT in nuclease-free water) and cells were used for encapsulation into lipid droplets using the Nadia instrument (Dolomite Bio, Hertfordshire, UK) based on manufacturer's guidelines. The conceptual principle is based on the DropSeq scRNA-seq approach. Library preparation including recovery of beads, reverse transcription, exonuclease treatment, and second strand synthesis procedures were followed as described previously (Emanuel W, Kirstin M, Vedran F, Asija D, Theresa GL, Roberto A, et al. Transcriptomic profiling of SARS-CoV-2 infected human cell lines identifies HSP90 as target for COVID-19 therapy. iScience. 2021;24(3):102151.). After incubation with second strand synthesis mix for 60 min at 37°C, beads were washed twice with 1 ml TE/0.01% Tween, once with 1 ml TE, and twice with water. cDNA amplification was achieved by dividing beads into 24 PCR reactions with SMART PCR mix in a total reaction volume of 50 μl using 4+9 cycle settings. After pooling 25 μl of each PCR reaction, the PCR products were cleaned-up using AMPure XP beads (Beckman Coulter). cDNA library quality and quantity were checked on a Bioanalyzer High Sensitivity Chip (Agilent) and the Qubit dsDNA HS Assay system (ThermoFisher). For each sample, 1000 pg of the cDNA library was used for Tagmenation using Nextera XT v2 DNA sample preparation (Illumina) including custom primers for amplification purposes. After PCR purification, quality and quantity assessment, paired-end sequencing was performed on a NextSeq500 (Illumina) and the NextSeq 500/550 High Output v2 Kit (75 cycles) (Read1CustSeqB custom primer for read 1 = 21 bp; read 2 = 63 bp).