alias: GSM1919743
title: GSM1919743: WT_23C_G6_rep2_mRNAseq; Caenorhabditis elegans; RNA-Seq
library_construction_protocol: Total RNA was extracted from frozen synchronized adult worm grinds (100-200 μl, pulverized in liquid nitrogen by grinding) using Trizol reagent (Life Technologies). mRNA was enriched using Poly(A)Purist MAG kit (Life Technologies). 0.5-1 μg of mRNA was used for each mRNA-seq library. DNA was removed from the RNA sample by DNase I treatment according to manufacture’s instruction (New England Biolabs).  RNA was then fragmented using Fragmentation Reagents (Ambion).  To prepare pre-mRNA libraries, fragmented RNA was treated T4 polynucleotide kinase (New England Biolabs).  The 3’ end of RNA was ligated to an adenylated DNA oligo  (IDT linker-1: 5’-rAppCTGTAGGCACCATCAATC-3’) using T4 RNA ligase 1 (New England Biolabs) without ATP. ~35-60 nt RNA captured by the 3’ linker oligo was purified using 8 % polyacrylamide (acrylamide:bis-acrylamide = 19:1) gels containing 8 M urea, followed by a 5’ end ligation reaction using a DNA-RNA hybrid oligo (5’-ACGCTCTTCCGATCTrNrNrNrN-3’, rNrNrNrN as 4-nt barcode) using T4 RNA ligase 1 (New England Biolabs) with ATP.  cDNA was synthesized by using SuperScript III Reverse transcriptase (Life Technologies) and primer SG460 (5’-GGAGTTCAGACGTGTGCTCTTCCGATCTATTGATGGTGCCTACAG-3’), followed by  PCR amplification with primers SG-465 (5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’) and SG-46X (5’-CAAGCAGAAGACGGCATACGAGAT[6-nt index]GTGACTGGAGTTCAGACGTGTGCTCTTCC-3’). To ensure that amplified DNA is still annealed to a true complement and avoid reannealing-distortion in the resulting libraries, PCR reactions were titrated with increasing cycle numbers; cycle numbers for which product levels have not saturated (i.e. product levels still able to increase substantially with additional cycles) were selected. After separating PCR products on 3 % agarose gels, DNA bands of the expected size were extracted (QIAchange® Gel Extraction Kit [Invitrogen]; omitting the 50°C heating step), followed by massive parallel DNA sequencing (Illumina HiSeq2000).
identifiers.namespace: GEO
identifiers.id: GSM1919743