RNA-Seq of FACS-Sorted Cells

RNA-Seq was performed as previously described^{19 (link)}. Briefly, RNA was isolated from 50,000 FACS-sorted cells using miRNeasy Mini Kit (QIAGEN). NEBNext Ultra RNA Library Prep Kit for Illumina was used to generate RNA-Seq libraries, following mRNA selection with Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). 12 cycles of index PCR was performed for multiplexing with NEBNext Multiplex Oligos for Illumina. The libraries were sequenced on a HiSeq 3000 in a 50-bp single read run.

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Yao C., Lou G., Sun H.W., Zhu Z., Sun Y., Chen Z., Chauss D., Moseman E.A., Cheng J., D’Antonio M.A., Shi W., Shi J., Kometani K., Kurosaki T., Wherry E.J., Afzali B., Gattinoni L., Zhu Y., McGavern D.B., O’Shea J.J., Schwartzberg P.L, & Wu T. (2021). BACH2 Enforces the Transcriptional and Epigenetic Programs of Stem-Like CD8+ T cells. Nature immunology, 22(3), 370-380.

Publication 2021

miRNeasy Mini Kit NEBNext Ultra RNA Library Prep Kit Poly(A) mRNA Magnetic Isolation Module NEBNext Multiplex Oligos

Cells Isolation Library Oligos Poly a mrna Rna seq

Corresponding Organization : National Institutes of Health

Other organizations : University of Colorado Anschutz Medical Campus, University of Pennsylvania, National Institute of Diabetes and Digestive and Kidney Diseases, Duke University, National Human Genome Research Institute, RIKEN Center for Integrative Medical Sciences, University of Regensburg, National Institute of Neurological Disorders and Stroke, National Institute of Allergy and Infectious Diseases

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independent variables

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control variables

50,000 FACS-sorted cells
MiRNeasy Mini Kit (QIAGEN) for RNA isolation
NEBNext Ultra RNA Library Prep Kit for Illumina for library generation
Poly(A) mRNA Magnetic Isolation Module (New England Biolabs) for mRNA selection
12 cycles of index PCR for multiplexing with NEBNext Multiplex Oligos for Illumina
HiSeq 3000 sequencing platform in a 50-bp single read run

controls

Not mentioned
Not mentioned

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