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_polzm_Polzm_TE_Not1TT_not5_17
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 polysome fraction was profiled henceforth was done as described in (Albanese et al., 2010).  Briefly , 400 ml yeast in exponential growth phase was treated with 100 ??g/ml cycloheximide, harvested, washed with cold lysis buffer, resuspended in 1 ml of buffer A (20 mM Hepes, 50 mM KCl, 10 mM MgCl2, 1% Triton X-100, 1 mM DTT, and protease inhibitor cocktail), and frozen as drops in liquid nitrogen. The cells were ground and 1 ml of buffer A was added to the powder, and the lysates were clarified by centrifugation at 14,000 g for 10 min. 20 OD lysate was applied on a 12 ml 7???47% sucrose gradient in buffer A and centrifuged in a rotor (SW41; Beckman Coulter) for 150 min at 39,000 rpm at 4??C. Fractions were collected using a UA/6 detector (ISCO, Inc.). When indicated, polysomes were dissociated by treatment with 25 mM EDTA. The fractions were TCA precipitated, separated by SDS-PAGE, and subjected to immunoblot analysis. 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: none
attributes.tag: Experimental Factor: genetic modification
attributes.value: gene knock out
attributes.tag: Experimental Factor: genotype
attributes.value: not5_Deletion
attributes.tag: Experimental Factor: strain
attributes.value: MAT_ leu2_20 ura3_met15_ lys2_ his3delta1 not1::NOT1-Tap-tag-URA3