GSM5591811: Mouse 1 somites; Mus musculus; OTHER
Embryos were placed in dissection media (DMEM/F-12 1:1, 10% FBS, and 1% penn-strep; GIBCO) and chilled on ice in a 60 mm petri dish (Falcon) until dissection. Each embryo was sequentially transferred to a small dissection dish with Sylgard bottom (Living Systems Instrumentation) in the same media, pinned facing down by small dissection pins (Living Systems Instrumentation), and the neural tube and somites were dissected out using two angled scissors (2.5 mm and 4 mm cutting edge; FST) from the first somite to the tail end. Prior to transferring the dissected somite/neural tube to a 1.5 ml RNase-free microfuge tube (ThermoFisher Scientific), any remaining ventral endoderm-derived tissue was removed. To the dissected somite/neural tube (~100 µl, after removing as much media as possible), 400 µl of Trizol reagent (Invitrogen) was added. The tube was vortexed until the tissue was dissolved, and then immediately frozen on dry ice and stored at -80 °C until RNA isolation. Total RNA was isolated using the Direct-zol RNA Miniprep kit (Zymogen), followed by quantification and analyses using Nanodrop One (ThermoFisher Scientific) and Bioanalyzer (Agilent), respectively, and stored at -80 °C until use. No-amplification non-tagging cap analysis of gene expression (nAnT-iCAGE) libraries were prepared using the cap-trap method as described in a detailed protocol (Murata et al., Methods Mol. Biol., 2014). In brief, 5 µg total mouse somite RNA was reverse transcribed (Superscript III, ThermoFisher) using random hexamer primers. Following cleanup with Agencourt RNAClean XP (Beckman Coulter), the RNA ends of RNA:cDNA hybrids were oxidized with NaIO4 (11.3 mM) on ice in the dark for 45 min, cleaned with Agencourt RNAClean XP, and biotinylated with biotin hydrazide (0.83 mM) at RT overnight. Single-stranded RNA was cleaved using RNAseONE ribonuclease and biotinylated RNA:cDNA hybrids were purified by incubation with Dynabeads M-270 streptavidin for 30 min, followed by washing and elution on a magnetic stand as previously described (Murata et al., Methods Mol. Biol., 2014)). Hybrid RNA was degraded with RNAseH, and any remaining free RNA was degraded with RNAse ONE. cDNA was concentrated in a centrifugal concentrator to ~5-10 µl and ligated to the pre-annealed double-stranded 5'linker containing a 6-nt barcode using ligation Mighty Mix at 16 °C for 16 hours. Following cDNA purification with Agencourt AMPure XP, cDNA was ligated to the pre-annealed 3'linker using ligation Mighty Mix at 16 °C for 16 hours. cDNA was treated with shrimp alkaline phosphatase and USER enzyme, followed by cleanup, second-strand synthesis using DeepVent DNA polymerase, primer degradation with Exonuclease I, and a final AMPure XP cleanup. Total cDNA was analyzed for quality and quantified using a Bioanalyzer (Agilent). DNA was pooled to a final concentration of 2 nM and sequenced on 2 lanes of a HiSeq FlowCell (Illumina) with 50 bp read length.