Illumina HiSeq 2000 paired end sequencing; 3' isoform specific RNA IP (3' isRIP) for PUF3 RBP in budding yeast
3' isoform specific RNA IP (3' isRIP) for PUF3 RBP in budding yeast
130814puf3_RIP_1
Grow single colony overnight in 5ml YPD at 30C unless the growth media is otherwise specified in sample annotations. Dilute in 50-100ml desired medium with starting OD600=0.1. Grow to OD600=1.0. Harvest cells for total RNA extraction. Flash frozen pellets of exponentially growing TAP tagged cells (OD600 0.5-0.8) were mechanically lysed with glass beads using a FastPrep24 agitator. An aliquot of each cell lysate was used as an input control. Cell lysates were subjected to immunoprecipitation with Dyna M280 sheep anti-rabbit IgG beads (Invitrogen) for 2h at 4??C. RNA-protein complex was released by TEV protease cleavage of the TAP-tag. The immunoprecipitated RNA was purified by phenol:chloroform:isoamyl alcohol extraction. In summary 10 ug of total RNA was fragmented, purified, retrotranscribed, end-repaired,  ligated, amplified and finally size selected.   Full protocol is describe in An efficient method for genome-wide polyadenylation site mapping and RNA quantification Wilkening et al., 2013