title: RNA sequencing of zebrafish:superficial epithelial cells 6dpf replicate
design: 100  150 larvae at 2 dpf, 6 dpf, 14 dpf, and 21 dpf were first rinsed with 1x DPBS (Gibco, 14190-144), then digested with collagenase (Sigma, C9891) and 0.25% trypsin-EDTA (Sigma, T4049). Digestion was stopped with DMEM (Gibco, 11995-065) with 10% NCS and rinsed with 1 x DPBS. Then, cells were suspended in DMEM-10% NCS and filtered with a 35-m cell strainer tube. Dissociated cells were stained with PI (1 g/ml for 106 cells per mL) for 5 min in the dark. All EGFP+ and PI- cells were sorted by FACSAria IIIu (BD Biosciences). Cells were directly sorted in buffer RLT supplemented with 1% -mercapto-ethanol provided in RNeasy Micro kit (Qiagen) and stored in -80 C until RNA extraction was performed. Total 2 replicates for each timepoint were collected. RNA extraction was carried out using RNeasy Micro kit according to manufacturers instructions. Sequencing was performed by the NGS High Throughput Genomics Core in Biodiversity Research Center, Academia Sinica, Taiwan using Illumina NextSeq2000 with paired-end 2 x 150 bp chemistry and a library selection of low-input stranded RNA library preparation, Poly-A.
library_name: LTS21-YW02