GSM2304919: mRNA-seq_set-32_OP50; Caenorhabditis elegans; RNA-Seq
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 fragmented by hydrolysis using Fragmentation Reagent Kit (Ambion). RNA was treated with T4 PNK (NEB). Then RNA 3’ end was ligated to IDT linker-1 (5’-rAppCTGTAGGCACCATCAATC-3’) using T4 RNA ligase 2, truncated (NEB, M0242S). RNA library of about 35-60 nt was size selected using 8 % polyacrylamide (acrylamide:bis-acrylamide = 19:1) gels containing 8 M urea (size selection also eliminated excess IDT linker-1). RNA 5’end was ligated to DNA-RNA hybrid oligo (5’-ACGCTCTTCCGATCTrNrNrNrN-3’, where rNrNrNrN is a 4-nt barcode) using T4 RNA ligase 1 (NEB) with ATP. First strand cDNA was synthesized using SuperScript III RT kit (Life Technologies) and primer SG-460 (5’-GGAGTTCAGACGTGTGCTCTTCCGATCTATTGATGGTGCCTACAG-3’). cDNA library was PCR amplified at early cycle (tested by different cycle number to capture linear amplification phase and avoid over amplification). PCR was done using a common primer SG-465 (5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’) and primer with 6-nt index for multiplexing (5’-CAAGCAGAAGACGGCATACGAGAT[ index]GTGACTGGAGTTCAGACGTGTGCTCTTCC-3’). Amplified library was size selected by 2% agarose gel and gel purified using QIAquick Gel Extraction Kit (QIAGEN).