GSM1356676: 10023X8_c.40_41del_RNA; Homo sapiens; RNA-Seq
Sections were lysed by 20 passages through a 26 gauge needle after addition of 500 μl of ice-cold 20 mM Tris*Cl pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% Triton X-100 and 100 μg / ml cycloheximide (Sigma).  For RNA-Seq, 100 μl of lysate was added to 300 μl of TriZOL LS reagent (Life Technologies) and total RNA was extracted according to the manufacturer’s specifications. For RPF-Seq, 6 μl of RNase I (100 U/ μl, Ambion) and 3 μl of Turbo DNase (2 U/ μl, Ambion) was added to 250 μl of lysate and incubated at room temperature with gentle agitation.  After 90 min., 750 μl of TriZOL LS reagent was added and ribonuclease-resistant RNA fragments were isolated by extraction.  RNA fragments were electrophoresed on a denaturing 15% polyacrylamide TBE-urea gel (Life Technologies, EC68852BOX). The gel region containing 26-38 nucleotide size RNA fragments was excised and RNA isolated by passive elution. Gel-purified RNA fragments were treated with 5 units of Antarctic Phosphatase (5 U/ μl NEB) in the presence of 40 units of RNase Inhibitor (40 U/ μl, Ambion) for 30 min. at 37oC, followed by 5 min. at 65oC to deactivate the enzyme.  Fragments were then treated with 20 units of T4 polynucleotide kinase (10 U/ μl NEB) for 60 min. at 37oC and purified using RNeasy MinElute columns (Qiagen) according to the manufacturer’s recommendations for small RNA. RPF-Seq libraries were built using the TruSeq Small RNA Sample Preparation Kit (Illumina) according to the manufacturer’s specifications, using Superscript II (Invitrogen).  After PCR to enrich for ligated fragments, the final gel purification was eliminated and instead the amplified library samples were cleaned using 50 μl of AMPure XP magnetic beads (Agencourt) according to the manufacturer’s specifications.   For RNA-Seq libraries, 4 μg of TRIzol-extracted total RNA from each sample was depleted for rRNA using Ribo-Zero rRNA Removal Kit Human/Mouse/Rat (Epicentre).  The rRNA-depleted total RNA was then processed using the TruSeq RNA Sample Preparation v2 Kit (Illumina) according to the manufacturer’s specifications using Superscript II (Invitrogen).