GSM8817785: mpra_F9_ds_mir155_rep2; Mus musculus; OTHER
MPRA: cells were lysed by addition of lysis buffer (10mM Tris-HCl pH7.5, 5mM MgCl2, 100mM KCl, 1% Triton X-100, 2mM DTT, 0.02U/uL SuperaseIn, 1 tab/10mL cOmplete mini EDTA-free protease inhibitor) then total RNA was extracted using Trizol-LS according to manufacturer instructions RBNS: AGO-miR-bound RNA was released from nitrocellulose membranes by proteinase K digestion according to manufacturer instructions, followed by phenol:chloroform extraction and ethanol precipitation MPRA and RBNS: extracted RNA was reverse-transcribed using SuperScript III reverse transcriptase according to manufacturer instructions using RT primers complementary to the 3ʹ constant region of the relevant RNA libraries Resultant library cDNA was then PCR-amplified using barcoded primers complementary to the 5ʹ and 3ʹ constant regions that also contained Illumina P5 and P7 constant region sequences for sequencing on an Illumina HiSeq 2500 (RBNS) or an Illumina NovaSeq 6000 (MPRA)