title: Illumina MiSeq paired end sequencing; Trancriptome-wide association of Not1and Polysomal occupancy in wt and not5 background
design: Trancriptome-wide association of Not1and Polysomal occupancy in wt and not5 background
library_name: ZIP_hSCrud_RIP_wt_2
library_construction_protocol: 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. The exponentially growing cells were harvested at OD 0.6-0.9and broken with glass beads. An aliquot of lysate was taken as Input control, the remaining was subjected to immunoprecipitation (IP) with Dyna M280 sheep anti rabbit IgG for 2h at 4??C and analyzed as described previously (Gupta et al., 2014). Negative control IP was performed following the above procedure on a BY4741 strain without a Tap-tag on any protein. In summary 10 ??g 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
attributes.tag: Experimental Factor: immunoprecipitate
attributes.value: IgG Against ProteinA in TAP tag
attributes.tag: Experimental Factor: genetic modification
attributes.value: none
attributes.tag: Experimental Factor: genotype
attributes.value: wild type genotype
attributes.tag: Experimental Factor: strain
attributes.value: MAT_ leu2_20 ura3_met15_ lys2_ his3delta1 not1::NOT1-Tap-tag-URA3