title: GSM6153478: AB7417; Danio rerio; RNA-Seq
library_construction_protocol: 5 pituitaries from each age group were dissected and pooled in 1ml ice-cold HEPES buffered saline (HBS), which was later changed to 250μl ice-cold PBS+/+ with prewarmed Liberase TM for 12 minutes at 30 degrees C. The tissues were then further dissociated with 750μl of TrypLE Express and 16μl DNaseI for 6 minutes at room temperature and stopped with 50μl FBS. Dissociated cells were pelleted at 500g for 5 minutes at 4 degree C, thereafter resuspended in 500μl ice-cold resuspension buffer (Leibovitz L-15 with 0.3mM Glutamine, penicillin 50 U/mL + streptomycin 0.05 mg/mL, FBS 1%, BSA 0.04%) and passed through a 40μm strainer. Cells were stained with propidium iodide then sorted into 384-well plates. The Mars-Seq single-cell library was prepared according to (Jaitin et al., 2014). In short, mRNA from the single cell was barcoded and converted to cDNA in the 384-well plate, then pooled and in vitro transcribed to RNA before fragmented into a library. The library was then ligated, reverse transcribed, and PCR amplified to attach the pool barcodes and Illumina sequencing codes. The library quality and concentration for each pool were checked before sequencing using an Illumina NextSeq 500 machine (Illumina, USA) at a median sequencing depth around 40,000 reads per cell.