title: GSM4490212: PEC_WT_1; Danio rerio; RNA-Seq
library_construction_protocol: Enteroendocrine cells (EECs) were isolated by dissecting the gut from about 200 Tg(pax6b:GFP)ulg515 larvae at 4 dpf, taking care of not including pancreatic tissue. Cell dissociation was next 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 two consecutive FACS purifications, the first in the “yield mode” and the second in “the purity mode”, using FACS Aria II. Four replicates of EEC containing about 3000 cells were prepared. Pancreatic endocrine cells (PECs) were also obtained from the Tg(pax6b:GFP)ulg515 line (Delporte et al., 2008b) by dissecting the dorsal pancreatic bud from about 200 27-hpf transgenic embryos. FACS selection was performed as described for EECs except that cell dissociation was performed in Tryple Select 1X ( Gibco) supplemented with 100 U/ml collagenase IV (Life Technologies) for 5 minutes. For the preparations of EECs and PECs from pax6b null mutant embryos, the pax6sa0086 line  (Verbruggen et al., 2010) was first crossed with the Tg(pax6b:GFP)ulg515 line; heterozygous pax6bsa0086 fish harboring the transgene (pax6b:GFP) were inbred to generate homozygous pax6bsa0086  transgenic embryos which were selected based on the absence or reduction of lens. The isolation of EECs and PECs from pax6bsa0086 homozygotes were performed in triplicates following the same procedure than for the wild-type larvae. The accuracy of pax6bsa0086 homozygotes selection was verified after the RNA-seq by checking the presence of the null sa0086 allele in 100% of pax6b reads in the mutant samples. Each EEC or PEC sample obtained after FACS 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 the Smart-seq2 protocol (Picelli et al., 2014). 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 60 millions of reads (100 base paired-ends).
attributes.tag: GEO Accession
attributes.value: GSM4490212