GSM3611939: T47D Charcoal Stripped Serum RNA-seq; Homo sapiens; RNA-Seq
Experiments were designed so as cells were ~80% confluent at the time of harvesting and lysis and at least 3 days after plating. Media was changed to fresh media in all flasks 24 hours prior to harvesting and to serum deprived media or media supplemented with charcoal stripped serum (T47D and ZR75-1 cells) 1 hour prior to harvesting as indicated. We used cycloheximide (final concentration 100 µg/mL, incubation for 10 minutes prior to collection) to halt the translating ribosomes. Ribosome profiling was conducted following the protocol from Ingolia, et al (Ingolia, N. T., Brar, G. A., Rouskin, S., McGeachy, A. M. & Weissman, J. S. The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments. Nat Protoc 7, 1534-1550, doi:10.1038/nprot.2012.086 (2012).) with the following modifications: (1) rRNA depletion was performed upfront using the Ribo-zero Gold rRNA removal kit (Illumina, cat# MRZG126) following manufacturer's instructions and (2) the linker-ligated ribosome protected fragments were retrieved using the RNA Clean and Concentrator Kit (Zymo Research, cat# R1015) following manufacturer's instructions. Total RNA (after digestion with Turbo DNase, Invitrogen cat# AM2238) and digested ribosome protected RNA was isolated using the Qiagen miRNeasy kit (Qiagen, cat# 217004) following manufacturer's protocol. Total RNA was rRNA depleted using the Ribo-zero Gold rRNA removal kit (Illumina, cat# MRZG126). RNA libraries were prepared for sequencing using standard Illumina protocols
GEO Accession
GSM3611939