Iso-Seq of Bos taurus: Dominette subcutaneous fat
The transcriptomes of tissues from the reference genome individual (L1 Dominette 01449) and testis from SuperBull 99375 were sequenced by the U.S. Meat Animal Research Center (USMARC, USDA-ARS, Clay Center, NE) using the PacBio RSII platform and supplied Iso-Seq protocol. Total RNA of each tissue was extracted using Trizol reagent (ThermoFisher Scientific) and the protocol included. Briefly, approximately 100 mg of tissue was ground in a mortar and pestle cooled with liquid nitrogen, and the powder was transferred to a tube with 1 ml of Trizol reagent added and mixed by vortexing. After 5 minutes at room temperature, 0.2 mL of chloroform was added and the mixture was shaken for 15 seconds and left to stand another 3 minutes. The tube was centrifuged at 12,000 x g for 15 minutes at 4ĄC. The RNA was precipitated from the aqueous phase with 0.5 mL of isopropanol. The RNA was further purified with extended DNase digestion to remove potential DNA contamination. The RNA quality was assessed with Fragment Analyzer (AATI, IA). Only RNA samples of RQN above 7.0 were used for library construction. PacBio IsoSeq libraries were constructed per the PacBio IsoSeq protocol. Briefly, starting with 3000 ng of total RNA, cDNA were synthesized by using SMARTer PCR cDNA Synthesis Kit (Clontech, CA). For the pooled, barcoded set, cDNA synthesis was performed separately for each with unique index adaptors as recommended in the IsoSeq protocol.  For all tissues, the cDNA was amplified using KAPA HiFi DNA Polymerase (KAPA Biotechnologies) for 10 or 12 cycles followed by bead-based purification.  For pooled/barcoded datasets, the amplification products for each library were quantified and pooled in equal amounts.  Individual or pooled amplified cDNA was then size selected into 4 fractions: 0.8-2 kb, 2-3 kb, 3-5 kb and > 5kb. The fragment size distribution was validated on a Fragment Analyzer (Advanced Analytical Technologies Inc, IA) and quantitated on a DS-11 FX fluorometer (DeNovix, DE). A second round of large scale PCR amplification was performed followed by enzymatic creation of blunt ends, after which SMARTbell adapters were ligated to the cDNA fragments.  Larger size fractions were given a second round of size selection to remove smaller molecules generated by the PCR, which tend to be preferentially loaded into the SMRTcells.  Each size fraction was sequenced on 4 or 5 SMARTcell v3 for single library samples, using P6-C4 chemistry and 4 or 6 hour movies (4 hour if the library was <3 kb average size, 6 hour if the library was >3 kb) on a PacBio RSII sequencer (Pacific Bioscience, CA).  The pooled/barcoded set was sequenced with only one SMRTcell for each of the two smaller size fractions, four cells for the 3-5 kb fraction, and five cells for the >5 kb fraction.
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