Illumina HiSeq 2000 paired end sequencing; RNA targets of YBX3 in Human HeLa cells
RNA targets of YBX3 in Human HeLa cells
IgG2_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. 125ul Dyna G magnetic beads were washed with lysis buffer 2x before coupling 10ug of anti-ZONAB (Bethyl Laboratories,  #A303-070A) or total rabbit IgG (Sigma) rotating for 1 hour at 4C, and free antibody was removed by washing the beads 2x with lysis buffer after coupling. RNAse/DNase treated extracts were added to the bead/antibody mixture and the YBX3-RNA complexes were immunoprecipitated for 2 h, 4C with gentle rotation. 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).