RNA Isolation and Sequencing from Mouse Tissues

RNA samples were isolated from fresh mouse tissues using RNA-Bee (TEL-TEST, Inc., Friendswood, TX, USA) or from formalin-fixed Paraffin Embedded (FFPE) slides using the Pinpoint Side RNA isolation Kit (Zymo Research, Cat R1007). RNA sequencing libraries were prepared with Kapa RNA HyperPrep Kit with RiboErase (Kapa Biosystems, Cat KR1351). Sequencing runs were performed on Illumina Hiseq 2500 in the single read mode of 51 cycles of read 1 and 7 cycles of index with V4 Kits. Real-time analysis (RTA) 2.2.38 software was used to process the image analysis and base calling. Reverse transcription was performed as described previously ^{36 (link)}, and RT-qPCR assays were carried out using SYBR GreenER qPCR Super Mix Universal (11762, Invitrogen) with specific primers (Supplemental Table 3) on the 7500 Real-Time PCR system (Thermo Fisher Scientific).

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He Y., Johnson D.T., Yang J.S., Wu H., You S., Yoon J., Lee D.H., Kim W.K., Aldahl J., Le V., Hooker E., Yu E.J., Geardts J., Cardiff R.D, & Sun Z. (2019). LOSS OF THE TUMOR SUPPRESSOR, TP53, ENHANCES THE ANDROGEN RECEPTOR MEDIATED ONCOGENIC TRANSFORMATION AND TUMOR DEVELOPMENT IN THE MOUSE PROSTATE. Oncogene, 38(38), 6507-6520.

Publication 2019

RNA-Bee Pinpoint Side RNA isolation Kit Kapa RNA HyperPrep Kit with RiboErase Hiseq 2500 SYBR GreenER qPCR Super Mix Universal 7500 Real-Time PCR system

Assays Embedded paraffin Formalin Isolation Mouse Primers Reverse transcription Sybr greener Tissues

Corresponding Organization :

Other organizations : City of Hope, Stanford University, Cedars-Sinai Medical Center, University of California, Davis

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Variable analysis

independent variables

RNA isolation method (RNA-Bee or Pinpoint Side RNA isolation Kit)

dependent variables

RNA sequencing data
Reverse transcription and RT-qPCR results

control variables

RNA sequencing library preparation method (Kapa RNA HyperPrep Kit with RiboErase)
Sequencing platform (Illumina Hiseq 2500)
Sequencing mode (single read, 51 cycles of read 1 and 7 cycles of index)
Image analysis and base calling software (RTA 2.2.38)
RT-qPCR assay (SYBR GreenER qPCR Super Mix Universal, 7500 Real-Time PCR system)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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