title: GSM5899258: Plate 9; Mus musculus; RNA-Seq
library_construction_protocol: RNA was extracted and reverse transcribed as previously published (PMID: 32194545; 32690951). In brief, single cells were FACS-sorted into ice-cold 96-well PCR plates (Thermofisher) containing 2µl lysis mix per well. Immediately after cell sorting, each plate was covered with an adhesive film (Thermofisher), briefly spun down in a benchtop plate centrifuge, and frozen on dry ice. The plates containing single cells in lysis mix were thawed on ice, briefly spun down in a benchtop plate centrifuge, and incubated in a thermal cycler for 3 minutes at 72°C (lid temperature 72°C). Immediately after, the plate was placed back on ice and prepared for the reverse transcription reaction, with SuperScript II (Thermofisher) in presence of RnaseOUT (Thermofisher), DTT (Thermofisher), betaine (Sigma-Aldrich), MgCl2 (Sigma-Aldrich), and well-specific template switching oligonucleotide. Plates with single-cell cDNA were stored at -20°C until further processing. For cDNA amplification, KAPA HiFi HotStart ReadyMix (Roche Diagnostics) was used with adapted primers. Libraries were prepared as previously published (PMID: 32194545; 32690951). In brief, amplified cDNA from each well of a 96-well plate were pooled, purified with two rounds of 0.6X solid-phase reversible immobilization beads (AmpureXP, Beckman, or CleanNGS, Proteigene) and finally eluted in nuclease free water. After tagmentation and neutralization, tagmented cDNA was amplified with Nextera PCR Mastermix containing Nextera i5 primer (Illumina), and custom i7 primer mix. Libraries generated from multiple 96-well plates of single cells and carrying distinct i7 barcodes were pooled for sequencing on an Illumina NextSeq2000 platform, with 100-cycles P2 flow cells, targeting 5 × 105 reads per cell in paired-end single-index mode with the following cycles: Read1 (Read1_SP, 67 cycles), Read i7 (i7_SP, 8 cycles), and Read2 (Read2_SP, 16 cycles).