GSM9700036: VSMCs,male,tdTomato; Mus musculus; RNA-Seq
The ascending aorta and aortic arch tissues were minced and digested in an enzyme solution containing DNase I (5 U/mL), collagenase IV (1 mg/mL), and dispase (1 mg/mL) in sequential 30-, 60-, and 90-minute steps, with trituration and cell counting performed between each step. After digestion, the cell suspension was passed through a 30-m cell strainer. After centrifugation, the supernatant was removed, red blood cells were lysed, and the cells were finally resuspended and counted to obtain a single-cell suspension. Next, the prepared single-cell suspensions were subjected to fluorescence-activated cell sorting (FACS) (BD, FACSAria III, U.S.A.) based on GFP and tdTomato fluorescence. Four groups were collected for downstream experiments: GFP-male, tdTomato-male, GFP-female, and tdTomato-female. The RNA-seq transcriptome library was prepared following SMART-Seq_V4 Ultra Low Input RNA Kit for Sequencing from Clontech (San Diego, CA) using 10ng of total RNA. Reverse transcription (one-strand synthesis) was performed first, and RNA with a polyA tail (major mRNA) was reverse transcribed using Oligo(dT) primer. Three cytosine (C) residues are added to the 3' end of the cDNA strand because of the use of a special active reverse transcriptase (MMLVRT) for reverse transcription. Two strands of cDNA were synthesized with the use of TSO (template-switching oligo) primers, thereby replacing RNA complementary to one strand of cDNA. Then, the cDNA was expanded to ng level by PCR amplification. DNA disruption using a modified, highly active Tn5 transposase was performed while the linker was added to both ends of the cDNA. After the last PCR amplification, the machine was ready for sequencing. After quantified by Qubit 4.0, paired-end RNA-seq sequencing library was sequenced with the NovaSeq X plus sequencer (2 × 150bp read length).