title: GSM4138513: HEAP miRNA library, Ago2Halo-LSL/+; p53fl/fl mice with Bcan-Ntrk1 glioma, biological rep 3; Mus musculus; OTHER
library_construction_protocol: For HEAP mRNA and miRNA libraries, RNAs were isolated along with the HaloTag-Ago2 fusion protein using HaloTag ligands conjugated to sepharose beads. RNAs were extracted from sepharose beads using phenol/chloroform. For input control libraries, RNAs were extracted from whole-cell lysates after RNAse A treatment using DynaBeads MyOne Silane. RNAs were ligated to RNA adaptors on both ends (3' RNA adaptor RL3: GUGUCAGUCACUUCCAGCGG; 5' RNA adaptor RL5D-6N: AGGGAGGACGAUGCGGNNNNNNG). After reverse transcription with a DNA primer DP3 (CCGCTGGAAGTGACTGACAC), the library was amplified with DNA primers DP3 and DP5 (AGGGAGGACGATGCGG). Illumina sequencing adaptors, sequencing primer binding sites and Illumina Truseq indexes for library multiplexing were introduced to the amplified libraries by PCR using primers DP3-barcode (CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCCCGCTGGAAGTGACTGACAC) and DSFP5 (AATGATACGGCGACCACCGACTATGGATACTTAGTCAGGGAGGACGATGCGG). After PicoGreen quantification and quality control by Agilent BioAnalyzer, libraries were run on a HiSeq 2500 in Rapid Mode in a 125/100bp single end run, using the HiSeq Rapid SBS Kit v2 (Illumina). Sequencing primers used were custom SSP1 primer (CTATGGATACTTAGTCAGGGAGGACGATGCGG) and the Illumina index read SP2 primer.
attributes.tag: GEO Accession
attributes.value: GSM4138513