High-multiplexed RNA-seq Library Prep

High-multiplexed library preparation for RNA-seq (PLATE-seq) was performed as described previously (19 (link)). Briefly, we captured poly-adenylated mRNA from cell lysates using a 96-well plate with oligo(dT) grafted to the inner walls of each well (Qiagen). Next, we eluted the poly-adenylated mRNA and reverse transcribed using 96 different barcoded oligo(dT) primers (Integrated DNA Technologies). Following exonuclease digestion of excess primers, the barcoded cDNA libraries were pooled for second-strand synthesis and Illumina library construction. We sequenced the resulting pooled and barcoded 3′-end libraries on an Illumina NextSEq. 500.

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Damaghi M., West J., Robertson-Tessi M., Xu L., Ferrall-Fairbanks M.C., Stewart P.A., Persi E., Fridley B.L., Altrock P.M., Gatenby R.A., Sims P.A., Anderson A.R, & Gillies R.J. (2021). The harsh microenvironment in early breast cancer selects for a Warburg phenotype. Proceedings of the National Academy of Sciences of the United States of America, 118(3), e2011342118.

Publication 2021

oligo(dT) barcoded oligo(dT) primers NextSEq. 500

Cell Digestion Exonuclease Library Mrna Oligo Poly Primers Rna seq Synthesis

Corresponding Organization : Moffitt Cancer Center

Other organizations : University of South Florida, National Center for Biotechnology Information, Columbia University Irving Medical Center

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Variable analysis

independent variables

Capturing poly-adenylated mRNA from cell lysates using a 96-well plate with oligo(dT) grafted to the inner walls of each well
Reverse transcribing the poly-adenylated mRNA using 96 different barcoded oligo(dT) primers

dependent variables

Sequencing the resulting pooled and barcoded 3′-end libraries on an Illumina NextSEq. 500

control variables

Exonuclease digestion of excess primers
Second-strand synthesis and Illumina library construction

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