GSM8305398: pituitary; Ctenopharyngodon idella; RNA-Seq
Tissues were washed in ice-cold Minimum Essential Medium and dissociated using trypsin, ethylene diamine tetraacetic acid（EDTA） and DNase II. After cell counting, fresh cells were washed twice in the NeuroGro medium (BasalMedia) and then resuspended at 1 106 cells per ml in PBS. Then added to the sample well of the SeekOne® DD Chip S3 (Chip S3). Following this, Barcoded Hydrogel Beads (BHBs) and partitioning oil were then added separately to their respective wells within Chip S3. Once emulsion droplets were generated, reverse transcription was performed at a temperature of 42℃ for 90 minutes, followed by inactivation at 85℃ for 5 minutes. The cDNA was then extracted from the broken droplets and amplified by PCR reactions. The resulting amplified cDNA product underwent a series of steps including purification, fragmentation, end repair, A-tailing, and ligation to sequencing adaptors. Finally, indexed PCR was performed to amplify the DNA, which represented the 3' polyA region of the expressing genes, including Cell Barcode and Unique Molecular Index. The indexed sequencing libraries were purified using SPRI beads and quantified by quantitative PCR (KAPA Biosystems KK4824). The libraries were then sequenced either on either an Illumina NovaSeq 6000 platform with PE150 read length or a DNBSEQ-T7 platform with PE150 read length.