Agilent rna screentape

For RNA extraction, cells were pelleted, frozen in liquid nitrogen, and stored at −80°C until RNA extraction. RNA extraction using Qiagen AllPrep DNA/RNA Mini Kit (Qiagen, Maryland, USA, Cat# 80204) per manufacturer’s instruction with cell homogenization in RLT buffer via QIAshredder (Qiagen, Maryland, USA, Cat# 79656). RNA quality control using Agilent Tapestation 2200 (Agilent, California, USA, Cat# G2964AA) and the Agilent RNA ScreenTape (Agilent, California, USA, Cat# 5067- 5576) and Agilent RNA ScreenTape Sample Buffer and Ladder (Agilent, California, USA, Cat# 5067- 5577, Cat# 5067- 5578) per manufacturer’s instruction. Library prep using Illumina RNA library prep kit (Illumina, California, USA, Cat # RS-122-2001) and sequenced on an Illumina HiSeq 4000 paired end using manufacturer’s instructions.
Sequencing reads aligned to Hg38 using HISAT2 (version 2.0.5), assembly and quantification was performed using StringTie (version 1.3.3) and differential expression was performed using R package Ballgown (version 2.6.0) as described (Pertea et al., 2015 (link), 2016 (link)). Exon skipping was determined using IGV Viewer Sashimi Plots (Robinson et al., 2011 (link)).

The retinal tissue sample from the affected female GR02 was collected directly in TRIzol Reagent (Invitrogen™, Carlsbad, CA, USA) for immediate RNA extraction. Retinal samples from three unaffected dogs—a beagle (BE02), a Labrador retriever (LR02), and a German shepherd (GS01)—were preserved in RNAlater (SigmaAldrich, Saint Louis, MO, USA) directly after euthanasia. The samples were then washed with 1 × PBS and between 50 to 100 mg of tissue was used for extraction. The samples from GR02, BE02, and GS01 were then homogenized in TRIzol Reagent with Precellys homogenizer (Bertin Instruments, Montigny-le-Bretonneux, France) and total RNA was extracted following the manufacturer’s instructions (Pub. No. MAN0001271, Rev. B.0.). Total RNA from LR02 was extracted with RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA concentration was measured using Qubit RNA BR Assay kit (Invitrogen™, Waltham, MA, USA). RNA integrity and quality were inspected with Agilent TapeStation 4150 with an Agilent RNA ScreenTape (Agilent Technologies, Santa Clara, CA, USA). PolyA-selection was carried out utilizing Dynabeads mRNA Purification Kit (Invitrogen™, Waltham, MA, USA), applying the manufacturer-provided protocol.

RNA extraction was done in the six cell lines at seven different time points: tissue‐derived myoblast and tissue‐derived myotube, as well as during hiPSC differentiation at Days 0, 3, 10, 17, and 25 (hiPSC‐derived myotube) using the miRNeasy Mini kit (217004, QIAgen) on the QIAcube instrument. RNAs coming from part A of the extraction protocol was used for mRNA‐seq and RT‐qPCR. RNAs coming from part B of the extraction protocol was used for miRseq. PartA RNA was quantified by Nanodrop spectrophotometer (ND‐1000, Thermo Fisher Scientific) and purity/quality (RIN ≥ 7) was assessed with the 2200 TapeStation using the Agilent RNA ScreenTape (5067‐5576/5067‐5577/5067‐5578, Agilent). PartB RNA was quantified and purity/quality was assessed with the 2100 Agilent Bioanalyzer using the Agilent small RNA kit (5067‐1548, Agilent).