GSM5782114: WT_2; Danio rerio; RNA-Seq
EECs were isolated by dissecting the gut from about 200 transgenic neurod1ΔUCE/ΔUCE or wild-type Tg(pax6b:GFP)ulg515 larvae at 4 dpf, taking care of not including pancreatic tissue.  Cell dissociation was performed by incubation in HBSS 1x supplemented with 100 U/ml collagenase IV and 0.3 U/ml Dispase (Life Technologies) for 10 minutes. Cells were washed in HBSS (Mg2+ and Ca2+ free) containing 1% BSA and GFP-expressing EECs were selected by FACS purification using FACS Aria II.  This procedure allows us to obtain between 1000 to 2000 isolated GFP+ cells.    Endodermal sox17-dsred+ cells were isolated by dissecting the trunk from about 150 double transgenic Tg(hsp70l:eGFP-2A-ARP/Ascl) or wild-type Tg(sox17:dsred) larvae at 52 dpf, heat-shocked at 38 and 48hpf, taking care of not including pancreatic tissue. Cell dissociation was performed by incubation in TrypLE select 1X (Gibco) for 8 minutes. Cells were washed in HBSS (Mg2+ and Ca2+ free) containing 1% BSA and dsred+ EECs were selected by FACS purifications using FACS Aria II.  This procedure allow us to obtain between 2000 to 4000 isolated dsred+ cells. Each FACS-sorted cells sample was directly pelleted by centrifugation and resuspended in 3.5 µl of reaction buffer, lysed by freezing in liquid nitrogen and stored at -80°C according the Smart-seq2 protocol [49]. cDNA was synthesised and amplified by a 13 cycles PCR reaction. Quality of cDNA was verified by 2100 High Sensitivity DNA assay (Agilent technologies). 1 ng cDNA was used for preparing each cDNA library using Nextera-XT kit (Illumina) and sequenced on Hi-seq 2000 to obtain around 20 million of reads (75 base single-end).