GSM3780694: MLE12 Untreated-2_seq; Mus musculus; ncRNA-Seq
Total RNA was extracted, labelled with P-32, run on acrilamide gel, band 15-100 bp in length were extracted and used for the library construction RNA was isolated from the two biological replicates of control and two of 10% CSE-treated MLE cells using Trizol (15596018, Ambion). The samples were treated with DNase I  (Sigma, 04716728001), re-isolated using Trizol, and RNA quality was checked using Agilent Bioanalyzer. The RNA was further treated with CIP (neb, M0290S), labeled with γ32P[ATP] using T4 PNK (neb, M0201S) and resolved on the 10% TBE-Urea polyacrylamide gels. 15-100 nt RNAs were isolated and used for the construction of libraries. 22nt 3'-DNA adaptor (/5Phos/NNNNCTGTAGGCACCATCAAT/3ddC) was ligated to the isolated RNAs using RNA-ligase T4 RNA-Ligase 2 (NEB, M0373S, RNL2 truncated KQ).  Each small RNA sample was reverse transcribed with a different, 102 nt long DNA primer(from RT-1 to RT-4). Each RT primer contained unique 3-nucleotide sample barcode (see "Reverse/- tag" and "Forward/+ tag" columns in the Characteristics field) to identify it after pooling and sequencing Paired-end sequencing was performed on an Illumina NextSeq platform (PE 2X 75 bp)