pU6 Lin28IR transfected NIH3T3 cells,  replicate 2
NIH3T3 cells were maintained in DMEM (Sigma) supplemented with 10 % fetal calf serum (Sigma), penicillin (100 U/mL, Invitrogen), and streptomycin (100 g/mL, Invitrogen) at 37 C and 5 % CO2 atmosphere. Cells were plated on 6-well plates and grown to 50 % density. Cells grown in a 24-well plate were transfected with 250 ng/well of U6-MosIR, U6-Lin28aIR, CAG-EGFP-MosIR, or CAG-EGFP-Lin28IR plasmids and cultured for 48 hours. Cells were washed with PBS, and total RNA was isolated using RNAzol (MRC) according to the manufacturers protocol. RNA quality was verified by Agilent 2100 Bioanalyzer. Libraries were constructed from total RNA using NEXTflex Small RNA-Seq Kit v3 (Bio Scientific) according to manufacturers protocol and sequenced on the Illumina HiSeq2000 platform at the Genomics Core Facility at EMBL.