Single cell RNA-sequencing of human melanoma: Sample 583_CD45pos_pre
cDNA for CD45+ single cells was generated using the Smart-Seq2 protocol as previously described. First, RNA was extracted using Agencourt RNAClean XP SPRI beads (Beckman Coulter, Cat A63987). The samples were then denatured at 72°C for 3 minutes, followed by reverse transcription using the Maxima H Minus Reverse Transcriptase enzyme (Thermo Fisher Scientific, Cat EP0753). Third, cDNA amplification was performed. Finally, two cDNA cleanup cycles were done using Agencourt AMPureXP SPRI beads (Beckman Coulter, Cat A63881) to discard any small-size fragments (e.g., primer dimers). Next, we generated libraries using the Nextera XT Library Prep kit (Illumina, Cat FC-131-1024). This step was followed by a cleanup process to eliminate any remaining primer dimers. Each library containing 384 CD45+ single cells was sequenced on a NextSeq 500 sequencer (Illumina) using the 75 cycles flow-cell/kit (Illumina, Cat 20024906), with paired-end, reads of 38 bases (for read1 and read2). Prior to and after library preparation, we checked each sample's quality by measuring their concentrations with the Qubit dsDNA high-sensitivity assay kit (Invitrogen, Cat  Q32854) and cDNA size distribution with the High-Sensitivity DNA Bioanalyzer Kit (Agilent, Cat 5067-4626).