Iso-Seq of intermuscular bones
A total amount of 5 g of total RNA (equally mixed with all RNAs) was used as the input material for library preparation. The mRNA containing polyA was enriched with Oligo(dT), which then was reversely transcribed into cDNA using the Clontech SMARTer PCR cDNA Synthesis Kit. After PCR amplification under optimal conditions, the collected cDNA fractions were treated with DNA damage repair, followed by end repair and ligation of SMRT adapters. Size fractionation and selection (< 4 kb and > 4 kb) were performed using the BluePippin Size Selection System (Sage Science, Beverly, MA). Two SMRT bell libraries were constructed with the Pacific Biosciences DNA Template Prep Kit 2.0 and SMRT sequencing was then performed on the Pacific Bioscience Sequel System.