RNA-seq Analysis of Mouse Blood

Blood was collected at baseline via cardiac puncture into K2 EDTA Plasma tubes (Sarstedt). Red blood cells were lysed with ACK lysis buffer, cells were washed, gently pelleted and resuspended in 500 μl of TRIzol (Invitrogen Life Technologies). RNA was extracted using TRIzol according to manufacturer’s instructions. rRNA was removed using the NEBNext rRNA Depletion Kit (NEB), followed by cDNA library preparation using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB). The libraries were sequenced on illumina HiSeq 3000 and the sequenced reads were aligned to the mouse genome using TopHat (65 (link), 66 (link)). Cufflinks and Cuffdiff were used to quantify transcripts and determine differential expression (65 (link), 66 (link)). Further analysis and visualization were performed using PARTEK and R.

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Rosshart S.P., Herz J., Vassallo B.G., Hunter A., Wall M.K., Badger J.H., McCulloch J.A., Anastasakis D.G., Sarshad A.A., Leonardi I., Collins N., Blatter J.A., Han S.J., Tamoutounour S., Potapova S., Foster St. Claire M.B., Yuan W., Sen S.K., Dreier M.S., Hild B., Hafner M., Wang D., Iliev I.D., Belkaid Y., Trinchieri G, & Rehermann B. (2019). Laboratory mice born to wild mice have natural microbiota and model human immune responses. Science (New York, N.Y.), 365(6452), eaaw4361.

Publication 2019

K2 EDTA Plasma tubes TRIzol NEBNext rRNA Depletion Kit HiSeq 3000

Blood Buffer Cardiac Cells Edta Genome Library Mouse Plasma Puncture Red blood cells Rrna Trizol

Corresponding Organization : University of Virginia

Other organizations : United States Department of Health and Human Services, National Cancer Institute, Center for Cancer Research, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Cornell University, National Institute of Allergy and Infectious Diseases, Washington University in St. Louis

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Transcript expression levels

control variables

Blood samples collected at baseline
Red blood cells lysed with ACK lysis buffer
Cells washed and gently pelleted
RNA extracted using TRIzol
RRNA removed using NEBNext rRNA Depletion Kit
CDNA library prepared using NEBNext Ultra RNA Library Prep Kit for Illumina
Libraries sequenced on Illumina HiSeq 3000
Sequenced reads aligned to mouse genome using TopHat

positive controls

None explicitly mentioned

negative controls

None explicitly mentioned

Annotations

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