RNA-seq Analysis of GFP+ Cells

Total RNA was isolated from green fluorescent protein (GFP)-positive, sorted cells using the RNeasy Mini Kit (Qiagen). RNA quality was checked using 2100 Bioanalyzer RNA 6000 Nanoassay (Agilent) or LabChip RNA Pico Sensitivity assay (PerkinElmer) before library generation. Libraries were prepared from total RNA with the TruSeq Stranded Total RNA Library Prep Kit (Illumina). Libraries were quantified using the Quant-iT PicoGreen dsDNA assay (Life Technologies) Kapa Library Quantification kit (Kapa Biosystems) or low pass sequencing on a MiSeq Nano v2 run (Illumina). One hundred cycle paired end sequencing was performed on an Illumina HiSeq 2500, HiSeq 4000, or NovaSeq 6000. RNA isolation, library preparation, and sequencing were performed on three biological replicates. RNA-seq data were mapped as described previously^{18 (link)} and HTSeq^{30 (link)} (version 0.6.1p1) were used to get gene-level count and estimated FPKM based on GENCODE (vM9)^{34 (link)}. Voom^{35 (link)} was used for gene differential expression analysis after trimmed mean normalization.

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Alexander T.B., Gu Z., Iacobucci I., Dickerson K., Choi J.K., Xu B., Payne-Turner D., Yoshihara H., Loh M.L., Horan J., Buldini B., Basso G., Elitzur S., de Haas V., Zwaan C.M., Yeoh A., Reinhardt D., Tomizawa D., Kiyokawa N., Lammens T., De Moerloose B., Catchpoole D., Hori H., Moorman A., Moore A.S., Hrusak O., Meshinchi S., Orgel E., Devidas M., Borowitz M., Wood B., Heerema N.A., Carrol A., Yang Y.L., Smith M.A., Davidsen T.M., Hermida L.C., Gesuwan P., Marra M.A., Ma Y., Mungall A.J., Moore R.A., Jones S.J., Valentine M., Janke L.J., Rubnitz J.E., Pui C.H., Ding L., Liu Y., Zhang J., Nichols K.E., Downing J.R., Cao X., Shi L., Pounds S., Newman S., Pei D., Guidry Auvil J.M., Gerhard D.S., Hunger S.P., Inaba H, & Mullighan C.G. (2018). The genetic basis and cell of origin of mixed phenotype acute leukaemia. Nature, 562(7727), 373-379.

Publication 2018

RNeasy Mini Kit 2100 Bioanalyzer RNA 6000 Nanoassay LabChip RNA Pico Sensitivity assay TruSeq Stranded Total RNA Library Prep Kit Quant-iT PicoGreen dsDNA assay Kapa Library Quantification kit MiSeq Nano v2 run HiSeq 2500 HiSeq 4000 NovaSeq 6000

Assay Biological Cells Dsdna Gene Gene expression analysis Green fluorescent protein Isolation Library Picogreen Rna seq Sensitivity

Corresponding Organization :

Other organizations : St. Jude Children's Research Hospital, UCSF Benioff Children's Hospital, Children's Healthcare of Atlanta, Emory University, University of Padua, Schneider Children's Medical Center, Tel Aviv University, Princess Máxima Center, National University of Singapore, Essen University Hospital, National Center For Child Health and Development, Ghent University Hospital, Children's Hospital at Westmead, Mie University, Newcastle University, University of Queensland, Charles University, Fred Hutch Cancer Center, Children's Hospital of Los Angeles, Children's Center, University of Florida, Johns Hopkins University, University of Washington, The Ohio State University, University of Alabama at Birmingham, National Taiwan University Hospital, National Cancer Institute, BC Cancer Agency, Canada's Michael Smith Genome Sciences Centre, Children's Hospital of Philadelphia, University of Pennsylvania

Top 5 similar protocols

Variable analysis

independent variables

Sorting cells based on green fluorescent protein (GFP) positivity

dependent variables

Gene expression levels (measured as gene-level counts and estimated FPKM)

control variables

RNA quality (checked using 2100 Bioanalyzer RNA 6000 Nanoassay or LabChip RNA Pico Sensitivity assay)
Library preparation (using TruSeq Stranded Total RNA Library Prep Kit)
Library quantification (using Quant-iT PicoGreen dsDNA assay, Kapa Library Quantification kit, or low pass sequencing on a MiSeq Nano v2 run)
Sequencing (100 cycle paired end sequencing on Illumina HiSeq 2500, HiSeq 4000, or NovaSeq 6000)
Biological replicates (3 replicates)

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