Data sets to process:

Positive control: SRP328042 Data are published in this article.
Experimental: SRP252265

To-Do:

• Obtain data in fastq format from Sequence Read Archive using fasterqdump options for paired end data - DONE
• Please data into directories named for the SRP number, e.g., SRP328042 and SRP252265 within a directory named "alt_splicing" under "nobackup" - DONE
• Make a note of the particular commands used to perform the data retrieval (see comment below)
• Create "samples" text file listing the SRR fastq files for running nf-core/rna-seq nextflow
• Run nf-core/rnaseq using proper maximum intron size parameter using "tomato.config"

Notes:

• Experimental datasets originally processed using code in https://bitbucket.org/hotpollen/rna-seq/src/master/ and https://bitbucket.org/hotpollen/flavonoid-rnaseq
• All Pollen project datasets are now in the SRA under the same project number ! (SRP252265)
• Ann is using a fork of flavonoid-rnaseq for all new code she's writing, on branch IGBF-3143. To find her fork, go to https://bitbucket.org/hotpollen/flavonoid-rnaseq and select "forks"
• Documentation for the pipeline we are using is here: https://nf-co.re/rnaseq/3.4/usage

Methods used to create positive control RNA-Seq data from SRP328042, according to the paper:

2.5.2. Preparation of RNA-Seq Library and Sequencing Total RNA was extracted utilizing Trizol reagent (Invitrogen, Waltham, MA, USA). RNA quantity and quality were determined by NanoDrop 1000 spectrophotometer (Thermo Scientific Inc., Waltham, MA, USA), 1% agarose gel electrophoresis and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Following the protocol described by [28], strand-specific RNA-Seq libraries from 3 biological replicates for each group from WW and DS anthers were prepared using 1 ng/µL of total RNA sample and sequenced by Novogene Biotech (Beijing, China) on Illumina HiSeq 4000 system (Illumina, Inc., San Diego, CA, USA) according to the manufacturer’s instructions. The raw sequence reads were deposited into NCBI Sequence Read Archive under accession the number PRJNA746070.

A PDF copy of the protocol paper (reference 28) for RNA-Seq library synthesis is attached.