title: NextSeq 500 sequencing; Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma
design: Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma
library_construction_protocol: Wildtype stain and drl:mCherry-positive zebrafish embryos were grown until tailbud stage and chorions were removed by incubating in 1 mg/mL Pronase (Roche), followed by washing in E3 medium. Around 300 embryos were used to generate the sample. The E3 medium was replaced by 1X PBS and the embryos were stored on ice until further processing. The embryos were dissociated using 2 mg/mL collagenase IV (Worthington) in DMEM (high glucose (4.5g/l) and NaHCO3, without L-glutamine and sodium pyruvate, Sigma-Aldrich) and incubated for 5 min in a water bath at 37oC. The embryonic tissues were triturated into a single-cell suspension by pipetting carefully up and down. When the embryos were not yet dissociated sufficiently, they were incubated for another 5 min. Cells were filtered through a 35 μm cell-strainer (Falcon,       round-bottom tubes with cell-strainer cap) and centrifuged at 6000 rpm for 30 sec. Cell pellets were resuspended in 1X HBSS (Gibco) containing 2% FBS and subjected to an additional round of centrifugation and resuspension. After washing, the cells were resuspended in 1X PBS.  drl:mCherry-positive cells were sorted using a FACS Aria III cell sorter (BD Bioscience). Cells were gated based on size and forward scattering, to exclude debris and doublets. The gating for the negative population was determined based on wildtype tailbud staged embryos. See also Figure S2 for gate setting. The SORTseq single-cell RNA-sequencing protocol was carried out as described previously (Muraro et al., 2016). Live mCherry-positive single cells were sorted in four 384-well plates (BioRad) containing 5 μl of CEL-Seq2 primer solution in mineral oil (24 bp polyT stretch, a 4 bp random molecular barcode (UMI), a cell-specific barcode, the 5′Illumina TruSeq small RNA kit adaptor and a T7 promoter), provided by Single Cell Discoveries. After sorting, the plates were immediately placed on ice and stored at −80°C.  In brief, ERCC Spike-in RNA (0.02 μL of 1:50000 dilution) was added to each well before cell lysis with heat shocking. Reverse transcription and second-strand synthesis reagents were dispensed using the Nanodrop II (GC biotech). After generation of cDNA from the original mRNA, all cells from one plate were pooled and the pooled sample was amplified linearly with in vitro transcription. The amplified RNA was then reverse transcribed to cDNA using a random hexamer primers (Hashimshony et al., 2016). To generate sequencing libraries, RPI-series index primers were used for library PCR.