title: Pacbio Long-read ISOseq on Sequel II of mouse cortex
design: 10ug of RNA from mouse cortex and striatum was treated with Terminator 5-Phosphate dependent exonuclease (Lucigen TER51020). cDNA was pre-amplified using KAPA HiFi HotStart Ready mix (Roche) for 27 cycles. Iso-seq libraries were prepares using the SMRTbell Template Express Prep Kit 2.0 (Pacific Biosciences) using 300 ng cDNA input. DNA library quality and consistency were verified with Qubit and Bioanalyzer and loaded onto the PacBio Sequel II sequencer. PacBio sequencing data was analyzed using SMRT Analysis software (Pacific Biosciences). Consensus reads were generated using ccs 4.2.0 with options --skip-polish --minLength 10 --minPasses 3 --min-rq 0.9 --min-snr 2.5. Adapters were removed with lima 1.11.0 with options --isoseq --min-score 0 --min-end-score 0 --min-signal-increase 10 --min-score-lead 0. Full-length non-chimeric reads were extracted, were oriented to the correct strand, and had poly-A tails clipped using isoseq3 refine 3.3.0 with options --min-polya-length 20 --require-polya. The resulting full-length non-chimeric reads were mapped to mm10 reference genome using minimap2 version 2.17-r941 (doi:10.1093/bioinformatics/bty191) with options -ax splice:hq -uf -MD .  TranscriptClean v2.0.2 (doi.org/10.1101/672931) was used for error correction with option --canonOnly. and TALON v5.0 (doi.org/10.1101/672931) was used for error correction and transcript identification and quantification. The module talon_label_reads with option --ar 20 was used to compute the fraction of As at the ends of read alignments. TALON databases of mouse (Ensembl 87 annotations) were created using talon_initialize_database with options --l 0 --5p 500 --3p 300.  The talon module was run with default parameters to identify transcripts using the initialized database.  Filtered transcript lists, which are limited to known and consistently observed transcripts, were generated using talon_filter_transcripts. Filtered and unfiltered transcript abundances were obtained using the talon_abundance module.
library_name: Male_LongRead_Cortex_NT_1