title: GSM5620297: tissue24h_1; Ambystoma mexicanum; RNA-Seq
library_construction_protocol: For each sample we pooled 5 pieces of tissue from 5 wildtype subadult axolotls. The tissues in liquid nitrogen with a mortar and pestle. A small quantity of bulk tissue powder was added to Trizol - this sample is labelled "bulk tissue" in the above data files. The powderized tissue was resuspended in lysis buffer (100 mM Tris pH7.5, 15 mM MgCl2, 150 mM NaCl, 1% Triton X-100, 2 mM DTT, 750U/mL SuperaseIn RNase Inhibitor (Ambion, AM2696), 0.2 mg/mL cycloheximide (Sigma-Aldrich, C7698-1G), 1X Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher, 78442), 25 U/mL Turbo DNase (Thermo Fisher, AM2238), 8% Glycerol, 0.5% DOC, 0.5 mg/mL heparin, 200 U/mL RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher, 10777019), 400U/mL RNasin (Promega, N2611)) at 500 μL per 70 mg of tissue powder. Samples were incubated on ice for 20 min with sporadic vortexing and dissociated by pipetting and passing through a 16G and 18G needle. The lysates were cleared and 75 μL were removed, combined with 1 mL of Trizol Reagent (Invitrogen, 15596018) and stored at -20°C (this was the "input" sample). The remaining lysate was loaded onto 4 mL sucrose gradients (17.5% - 50%) and underwent centrifugation for 2.5 h at 4°C at 35,000 rpm on Beckman SW60 rotor. Gradients were fractionated using a density gradient fraction system (Brandel, BR-188). Fractions were collected into 100 μL of 10% SDS and subjected to acid-phenol mRNA extraction (for "free/RNP", "light" or "heavy" polysome fraction samples) or Trizol extraction ("input" or "bulk tissue" samples). Pellets were precipitated in isopropanol, washed in 75% ethanol and resuspended in 10 μL (for fractions) or 40 μL (for input) of Ultra Pure DNase/RNase-Free Distilled Water (Invitrogen, 10977-015). The fractions were pooled as follows: : (1) RNP to 40S, (2) “light” polysome containing the monosome and disome fractions, (3) “heavy” polysome containing all fractions with 3+ ribosomes. The total volume of the fraction pools was topped up to 180 μL with Ultra Pure water and 1 μL of 1:200 ERCC RNA Spike-In Mix (Invitrogen, 4456740) was added to each fraction pool. The samples underwent DNase treatment with TURBO DNase (Ambion, AM2238) for 30 min at 37°C in a total volume of 500 μL followed by acid-phenol mRNA extraction. The samples were then resuspended in 30 μL of Ultra Pure DNase/RNase-Free Distilled Water. Libraries were prepared by the Stanford Genomic Services Center using the TruSeq Stranded Total RNA Library Prep with Ribo-Zero Gold Human/Mouse/Rat (Illumina) and sequenced (PEx150) on a HiSeq 4000 (Illumina) following the manufacturer's instructions.