GSM9756264: HashTag; Mus musculus; RNA-Seq
BD Rhapsody system Single-cell suspensions of TSCs and D-TSCs were loaded onto the BD Rhapsody system using the BD Rhapsody Targeted & AbSeq Reagent Kit (BD Biosciences) according to the manufacturer's instructions. Individual cells were captured in microwells and lysed in the presence of barcoded magnetic beads, enabling cell-specific transcript labeling via unique molecular barcodes. Captured RNA on BD Rhapsody magnetic beads was reverse transcribed, followed by Exonuclease I treatment for 60 min. The resulting beads were subjected to the TAS-Seq workflow for cDNA and hashtag library preparation (Shichino et al. 2022). Briefly, reverse-transcribed beads were treated with terminal deoxynucleotidyl transferase (TdT; P7070L, Enzymatics) and RNase H (Y9220L, Enzymatics), followed by second-strand cDNA synthesis and the first round of whole-transcriptome amplification. Amplified cDNA was purified using 0.65× AMPure XP beads (Beckman Coulter), while hashtag oligonucleotides were recovered from the unbound fraction by an additional 0.65× AMPure XP bead purification. cDNA and hashtag libraries were further amplified by a second round of WTA and hashtag amplification, respectively, followed by purification. Amplified libraries were fragmented and subjected to adaptor ligation and index PCR using the NEBNext Ultra II FS Library Prep Kit for Illumina (New England Biolabs) to generate sequencing-ready libraries.