title: GSM4760209: KPC_ACVR2B/Fc_Late [75]; Mus musculus; RNA-Seq
library_construction_protocol: RNA extraction and purification from tissue snap frozen in liquid nitrogen was done via homogenization in Trizol and the Qiagen miRNeasy Mini Kit. The Illumina TruSeq Standed mRNA standard methods were used for RNA sequencing (RNAseq). Total RNA extracted from the mouse quadriceps skeletal muscles was first evaluated for its quantity, and quality, using Agilent Bioanalyzer 2100. For RNA quality, a RIN number (RNA Integrity Number) of 7 or higher is desired. Five hundred nanograms of total RNA was used for library preparation. Briefly, cDNA library preparation included mRNA enrichment/purification, RNA fragmentation, cDNA synthesis, ligation of index adaptors, and amplification, following the TruSeq Stranded mRNA Sample Preparation Guide, RS-122-9004DOC, Part# 15031047 Rev. E (Illumina, Inc.). Each resulting indexed library with average insert size of 150-200b was quantified and its quality accessed by Qubit and Agilent Bioanalyzer, and multiple libraries pooled in equal molarity. Five microliters of 2 nM pooled libraries per lane were then denatured, neutralized and applied to the cBot for flow cell deposition and cluster amplification, before loading to HiSeq 4000 for 75b paired-end read sequencing (Illumina, Inc.). Approximately 30M reads per library were generated. A Phred quality score (Q score) was used to measure the quality of sequencing. More than 90% of the sequencing reads reached Q30 (99.9% base call accuracy).