GSM7988809: 4th round of regeneration; Danio rerio; RNA-Seq
For each experiment about 8000 cells from zebrafish fin regenerates were flow-sorted into BSA-coated PCR tubes containing 1 µl of PBS with 0.04 % BSA. All cells were carefully mixed with reverse transcription mix before loading them in a Chromium Single Cell A Chip on the 10X Genomics Chromium controller and processed further following the guidelines of the 10X Genomics user manual for single cell 3' RNA-seq v2. In short, the droplets were directly subjected to reverse transcription, the emulsion was broken and cDNA was purified using silane beads. After amplification of cDNA with 12 cycles, it underwent a purification with 0.6 volume of SPRI select beads. After quality check and quantification using the Fragment Analyzer (Agilent), 30 ng cDNA were used to prepare sc RNA-seq libraries - involving fragmentation, dA-Tailing, adapter ligation and a 12 cycles indexing PCR based on manufacturer's guidelines. After quantification, both libraries were sequenced on an Illumina Nextseq500 system in paired-end mode with 26 bp/57 bp (for read 1 and 2 respectively), thus generating ~60-90 mio. fragments for the transcriptome library on average.