Illumina HiSeq 2000 paired end sequencing; RNA targets of YBX3 in Human HeLa cells
RNA targets of YBX3 in Human HeLa cells
SMI2_p
HeLa cells were placed on ice, washed 3x in PBS, subjected to 254 nm UV-crosslinking (150 mJ/cm2), and lysed on the plate in lysis buffer (500mM NaCl, 50mM Tris-HCl pH 7.5, 8.5% glycerol, 1mM EDTA pH 8.0, 0.5% Triton X100, 2mM BME and complete EDTA free protease inhibitor (Roche)). Lysates were sonicated (Branson sonifier) and cleared by centrifugation at 16 000 x g for 10 min at 4C.  Lysates were treated with RNase I (10ul 1:25 dilution/extracts from 8.0 x E6 cells; Ambion) to fragment RNA and turbo DNase (2ul/extracts from 8.0x E6 cells, Thermo) for 5minutes at 37C and 1200 rpm shaking. Samples processed for all subsequent steps following supplementary protocol one described previously (Van Nostrand et al. 2016 PMID: 27018577). Standard eCLIP library preparation was done following supplementary protocol one described previously (Van Nostrand et al. 2016 PMID: 27018577). The SMI samples were amplified using 9 cycles while the IP samples were amplified using 18 cycles. The samples were multiplexed and sequenced on Illumina HiSeq2000 platform (125PE v4 chemistry).