RNA-seq Library Preparation Protocols

For infant control and CDM samples, RNA-seq libraries were prepared using the SMART-Seq version 4 ultralow input RNA kit (Clontech) per the manufacturer's instructions and sequenced using an Illumina HiSeq 2000. For PolyA-seq libraries, total RNA (130 ng) was used as a starting template, and libraries were generated as described (Batra et al. 2014 (link)) with the modification of adding barcodes to the library amplification primers to accommodate multiplex sequencing. For mouse P0 muscle and primary myoblast samples, RNA was isolated using the Direct-Zol RNA miniprep kit (Zymo Research). RNA-seq libraries were prepared from total RNA (500–700 ng) using the stranded RNA-seq kit with RiboErase (Kapa Biosystems) per the manufacturer's protocol. The CDM PolyA-seq and all mouse libraries were sequenced using an Illumina NextSeq 500.

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Thomas J.D., Sznajder Ł.J., Bardhi O., Aslam F.N., Anastasiadis Z.P., Scotti M.M., Nishino I., Nakamori M., Wang E.T, & Swanson M.S. (2017). Disrupted prenatal RNA processing and myogenesis in congenital myotonic dystrophy. Genes & Development, 31(11), 1122-1133.

Publication 2017

SMART-Seq version 4 ultralow input RNA kit HiSeq 2000 Direct-Zol RNA miniprep kit RiboErase NextSeq 500

Infant Library Mouse Muscle Myoblast Polya Primers Rna seq

Corresponding Organization :

Other organizations : University of Florida, Osaka University, National Center of Neurology and Psychiatry

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Variable analysis

independent variables

RNA-seq library preparation method (SMART-Seq version 4 ultralow input RNA kit, PolyA-seq library generation as described in Batra et al. 2014, stranded RNA-seq kit with RiboErase)
Sample type (infant control, CDM, mouse P0 muscle, primary myoblast)

dependent variables

Gene expression measured by RNA-seq

control variables

RNA input amount (130 ng for PolyA-seq, 500-700 ng for mouse samples)
Sequencing platform (Illumina HiSeq 2000, Illumina NextSeq 500)

controls

No positive or negative controls were explicitly mentioned.

Annotations

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