GSM8999056: zebrafish general cell population- control, replicate1; Danio rerio; RNA-Seq
RNA isolation. Purification of RNA from sorted cells (~20,000 iridophores or general cell population) was done using Dynabeads® mRNA DIRECT™ Purification Kit (Ambion, 61012) according to manufacturer protocol. The purified RNA samples were frozen at -80°C until the step of RNA-seq library preparation. Bulk MARS-Seq preparation. RNA-seq libraries were prepared at the Crown Genomics Institute of the Nancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science. A bulk adaptation of the MARS-Seq (Jaitin, D. A. et al.  Science 343, 776-779 (2014) and  Keren-Shaul, H. et al. Nat Protoc 14, 1841-1862 (2019)  protocol was used to generate RNA-Seq libraries for expression profiling of iridophores and general control cell population. Briefly, RNA (all RNA amount purified from ~20,000 sorted cells) from each sample was barcoded during reverse transcription and pooled. Following Agencourct Ampure XP beads cleanup (Beckman Coulter), the pooled samples underwent second-strand synthesis and were linearly amplified by T7 in vitro transcription. The resulting RNA was fragmented and converted into a sequencing-ready library by tagging the samples with Illumina sequences during ligation, RT, and PCR.  Libraries were quantified by Qubit and TapeStation as well as by qPCR as previously described (Jaitin, D. A. et al.  Science 343, 776-779 (2014) and  Keren-Shaul, H. et al. Nat Protoc 14, 1841-1862 (2019)  For Danio rerio, the housekeeping gene actinb1 was selected for its relatively high and uniform expression and was specifically calibrated for the MARS-Seq protocol. Sequencing was done on a NextSeq 500 using NextSeq High Output, 75 cycles, allocating 400M reads in total (Illumina).