MiRNomics reveals 3D cultures better mimic tumors in vivo
Total RNA was used as starting material. 3’ adapters were ligated to the specific 3’OH group of micro RNAs followed by ligation  of 5’ adapter. Adapter ligated fragments were reverse transcribed with Superscript III Reverse transcriptase (Invitrogen, Whitefield, Bangalore) by priming with Reverse transcription primers. cDNA thus formed was enriched and barcoded by PCR amplification (15 cycles). Amplified library was size selected using Polyacrylamide gel. Library was size selected in the range of 140 bp– 180 bp followed by overnight gel elution and salt precipitation in presence of Glycogen (Invitrogen, Whitefield, Bangalore), 3M Sodium Acetate (Sigma, Saint Louis, Missouri, USA) and Absolute Ethanol (Tedia Company, Tedia Way, Fairfield,USA). Pellet was re-suspended in Nuclease free water (Invitrogen, Whitefield, Bangalore). Illumina-compatible sequencing library was initially quantified by Qubit fluorometer (Thermo Fisher Scientific, MA, USA) and its fragment size distribution was analyzed on Agilent TapeStation