TY - JOUR
T1 - Cell-type specific polysome profiling from mammalian tissues
AU - Seimetz, Joseph
AU - Arif, Waqar
AU - Bangru, Sushant
AU - Hernaez, Mikel
AU - Kalsotra, Auinash
N1 - Funding Information:
This work was supported by NIH ( R01HL126845 ) grant to A.K., NIH pre-doctoral NRSA fellowship ( F30DK108567 ) to W.A, and NIH Tissue Microenvironment training program ( T32-EB019944 ) to S.B. J.S. was partly supported by NIH Chemistry–Biology Interface training program ( T32-GM070421 ) and American Heart Association pre-doctoral fellowship ( 17PRE33670030 ).
Publisher Copyright:
© 2018 Elsevier Inc.
PY - 2019/2/15
Y1 - 2019/2/15
N2 - The regulation of gene expression occurs through complex relationships between transcription, processing, turnover, and translation, which are only beginning to be elucidated. We know that at least for certain messenger (m) RNAs, processing, modifications, and sequence elements can greatly influence their translational output through recognition by translation and turn-over machinery. Recently, we and others have combined high-throughput sequencing technologies with traditional biochemical methods of studying translation to extend our understanding of these relationships. Additionally, there is growing importance given to how these processes may be regulated across varied cell types as a means to achieve tissue-specific expression of proteins. Here, we provide an in-depth methodology for polysome profiling to dissect the composition of mRNAs and proteins that make up the translatome from both whole tissues and a specific cell type isolated from mammalian tissue. Also, we provide a detailed computational workflow for the analysis of the next-generation sequencing data generated from these experiments.
AB - The regulation of gene expression occurs through complex relationships between transcription, processing, turnover, and translation, which are only beginning to be elucidated. We know that at least for certain messenger (m) RNAs, processing, modifications, and sequence elements can greatly influence their translational output through recognition by translation and turn-over machinery. Recently, we and others have combined high-throughput sequencing technologies with traditional biochemical methods of studying translation to extend our understanding of these relationships. Additionally, there is growing importance given to how these processes may be regulated across varied cell types as a means to achieve tissue-specific expression of proteins. Here, we provide an in-depth methodology for polysome profiling to dissect the composition of mRNAs and proteins that make up the translatome from both whole tissues and a specific cell type isolated from mammalian tissue. Also, we provide a detailed computational workflow for the analysis of the next-generation sequencing data generated from these experiments.
KW - Cell-type isolation from tissues
KW - Next-generation sequencing and bioinformatics
KW - Polysome profiling
KW - Ribosomal occupancy shift
KW - Translation regulation
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U2 - 10.1016/j.ymeth.2018.11.015
DO - 10.1016/j.ymeth.2018.11.015
M3 - Article
C2 - 30500367
AN - SCOPUS:85058232662
SN - 0076-6879
VL - 155
SP - 131
EP - 139
JO - Methods in Enzymology
JF - Methods in Enzymology
ER -