title: GSM6722927: Zebrafish Muscle Illumina; Danio rerio; RNA-Seq
library_construction_protocol: Tissues were put in a Lysing Matrix D tube placed on ice. Then 600 µL of lysis buffer were added. The tubes were firmly closed and agitated for 2×60 s at speed 6 m/s on a FastPrep-24 homogenizer. Total RNA was then extracted using the Agencourt RNAdvance Tissue Kit following the provider's recommendations. Total RNA was quantified using the Quant-iT™ RiboGreen™ RNA Assay Kit on a Spectramax M2. Total RNA quality was assessed using Fragment Analyzer-96 with DNF-471-0500 Standard Sensitivity RNA Analysis Kit. RNA-Seq:  An amount of 500 ng of total RNA was used for each sample. A bead-based mRNA capture followed by a fragmentation, first and second strand synthesis, an end repair, and a 3`adenylation and indexed linker ligation were performed. The adapter ligated cDNA library fragments were then enriched by pPolymerase chain reaction (PCR) using 15 cycles of amplification (Illumina TruSeq Stranded mRNA protocol). Samples were then bead purified, pooled equimolar Iso-Seq:  An amount of 300 ng of total RNA was used to prepare the Iso-Seq libraries for PacBio following exactly the Iso-Seq Express Template Preparation for Sequel and Sequel II Systems version 02 (October 2019). Libraries sizes were assessed using a FemtoPulse with FP-1002-0275 gDNA 165kb Analysis Kit. Libraries quantifications were performed using a Qubit fluorometer with Q32854 Qubit dsDNA HS Assay Kit (500×).