title: GSM5270963: fxr -/- zebrafish intestinal epithelial cells; Danio rerio; RNA-Seq
library_construction_protocol: Larvae zebrafish were dissociated using standard protocol, after which the fxr+/+; TgBAC(cldn15la-GFP) and fxr-/-; TgBAC(cldn15la-GFP) cells were subjected to FACS at the Duke Cancer Institute Flow Cytometry Shared Resource and were sorted side by side with two identical Beckman Coulter Astrios instruments. Non-transgenic and single transgenic controls (pools of 50 fish/genotype) were prepared as above and used for gating and compensation. Approximately 120 k GFP positive 7-AAD negative cells per genotype were collected in 1.5 mL of DMEM/F12 supplemented with 10% heat-inactivated FBS and 10 μM Y-27632 ROCK1 inhibitor and were immediately subjected to the library construction. Each single-cell RNA sequencing library was generated from 10,000 FACS sorted TgBAC(cldn15la-GFP) IECs of the indicated genotype following the 10x Genomics Single Cell 3' protocol by the Duke Molecular Genomics Core. The sequencing ready libraries were cleaned with both Silane Dynabeads and SPRI beads, and quality controlled for size distribution and yield with the Agilent D5000 screenTape assays using the Agilent 4200 TapeStation system. Illumina P5 and P7 sequences, a sample index, and TruSeq read 2 primer sequence were ligated for Illumina bridge amplification. Sequence was generated using paired-end sequencing on the Novaseq SP flow cell sequencing platform at a minimum of 40 k reads/cell.