title: IsoSeq of Sus scrofa: juvenile  castrated male spleen
design: Sequencing the transcriptome of nine porcine tissues by the PacBio IsoSeq and Illumina RNA-seq technologies:The transcriptome of three tissues (liver, spleen and thymus) from a single cross-bred pig, from which the PacBio long read-based reference genome was assembled by using DNA of the lung tissue, was sequenced by the Meat Animal Research Center (MARC, USDA, Clay Center, NE) using the Illumina NextSeq500 and PacBio RSII platforms for RNAseq and IsoSeq, respectively. Total RNA of each of the threes tissues were 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 4°C.  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) according to the IsoSeq protocol (Pacific Biosciences, CA). Then the cDNA was amplified using KAPA HiFi DNA Polymerase (KAPA Biotechnologies) for 10 or 12 cycles followed by purification and size selection 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). After a second round of large scale PCR amplification and end repair, SMARTbell adapters were separately ligated to the cDNA fragments.  Each size fraction was sequenced on 4 or 5 SMART Cell v3 using P6-C4 chemistry and 6 hour movies on a PacBio RS II sequencer (Pacific Bioscience, CA).   Short read RNAseq libraries were prepared using TruSeq stranded mRNA LT kits and supplied protocol (Illumina, CA), and sequenced on the NextSeq500 platform using 2x75 paired end reads and v2 sequencing chemistry.     Error-correction and redundancy reduction of PacBio IsoSeq full-length cDNA reads:The Read of Insert (ROI) were determined by using consensustools.sh in the SMRT-Analysis pipeline v2.0, with reads which were shorter than 300 bp and whose predicted accuracy was lower than 75% being removed. Full-length, non-chimeric cDNA reads were identified by running classify.py. Primer sequences as well as the poly(A) tails were trimmed off prior to further analysis. Paired-end Illumina RNA-seq reads from the same tissue samples were trimmed to remove the adaptor sequences and low-quality bases using Trimmomatic [2] with explicit option settings: ILLUMINACLIP:adapters.fa:2:30:10:1:true LEADING:3 TRAILING:3  SLIDINGWINDOW:4:20 LEADING:3 TRAILING:3 MINLEN:25, and overlapping paired-end reads were merged using PEAR [3]. Subsequently, the merged and unmerged RNA-seq reads from the same tissue samples were treated as single end reads and in silico normalized with a Trinity untility, insilico_read_normalization.pl, with the following settings:  --max_cov 50 --max_pct_stdev  100 --single. Errors in the full-length, non-chimeric cDNA reads were corrected with the preprocessed RNA-seq reads from the same tissue samples by using proovread (v2.12) [4] . Untrimmed sequences with at least some regions of high accuracy in the .trimmed.fq files were extracted based on sequence IDs in .untrimmed.fa files to balance off the contiguity and accuracy of the fi