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Sample GSM6758376

Query DataSets for GSM6758376

Status

Public on Jul 04, 2023

Title

PDB_25°C_Iso-Seq

Sample type

SRA

Source name

mycelium

Organism

Zymoseptoria tritici

Characteristics

tissue: mycelium
strain: IPO323
growth media: PDB

Growth protocol

The reference isolate of Z. tritici IPO323 was stored at -80°C as a yeast-like cell suspension (10e7-8 cells/mL in 30% glycerol). Z. tritici was grown at 18°C in the dark on Yeast extract Peptone Dextrose (YPD) agar, Potato Dextrose Broth (PDB). For RNA production, Z. tritici IPO323 isolate (4 days old yeast-like cells diluted to 10-5 cells/mL final) was cultivated in 75 mL agitated liquid cultures (500 mL Erlen flasks, 150 rpm) at 18°C in the dark for 4 days. Different media were used including Glucose-NO3 synthetic medium defined as MM-Zt (E. Marchegiani et al, 2015). MM-Zt was modified by replacing glucose (10 g/L) by different carbon sources (Xylose, Mannitol, Galactose, Sucrose at 10 g/L)). Histone Deacetylase inhibitors such as trichostatin (TSA, Sigma ref, 1 microM final) and SAHA (SAHA, Sigma ref, 1 mM final) were added to MM-Zt. The composition of complex media (YPD, PDB, AE) was already described (G. Scalliet et al, 2012). Cultures of IPO323 in YPD and PDB were performed at 18°C and 25°C, while AE cultures were performed only at 18°C. A total of 14 culture conditions were used for RNA production. All cultures for RNA-Seq were performed in triplicates.

Extracted molecule

polyA RNA

Extraction protocol

Cultures were centrifuged at 3000 rpm for 10 minutes and mycelium pellet washed with water was frozen with liquid nitrogen. Frozen mycelium was lyophilized and kept at -80°C until extraction. RNAs were extracted using Quiagen Plant RNAeasy Kit according to manufacturer protocol (Ref. 74904, Quiagen France SAS, Courtaboeuf, France).
PacBio Iso-Seq libraries preparation and sequencing was performed by the INRAE platform Gentyane (http://gentyane.clermont.inrae.fr). SMARTer PCR cDNA Synthesis Kit (ref 634926, Clontech, Mountain View, CA, USA) was used for polyA primed first cDNA strand synthesis followed by optimized PCR amplification and library preparation using SMRTbell Template Prep Kit (ref 101-357-000, Pacific Bioscience, Menlo Park, CA, USA) according to manufacturer protocols. The cDNA libraries were prepared without size selection and bar-coded for multiplexing. Sequencing was performed on a PacBio SEQUEL (version 1). Illumina RNA-seq single stranded libraries were prepared using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490, New England BioLabs, Ipswich, Massachusetts, USA) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB #E7765, New England BioLabs, Ipswich, Massachusetts, USA). Custom 8 bp barcode was added to each library during the preparation process. Sample were pooled and cleaned with magnetic beads included in the library preparation kit. The pool was run on a lane of Illumina HiSeqX (Illumina, San Diego, California, USA) using a 150-cycle paired end run

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Sequel

Description

isoforms.ranking.gff.gz

Data processing

Iso-Seq raw data were processed with the Iso-Seq V3.2 pipeline from PacBio providing polished Circular Consensus Sequences (CCS). CCS were then mapped to the Z. tritici IPO323 genome with Gmap (2019-01- 31) and unmapped, low mapping quality (≤0) or multi-mapped CCS were filtered out.
RNA-Seq data were cleaned and trimmed with Trimomatic (v 0.36). The cleaned sequences were then mapped to Z.tritici IPO323 genome using STAR (v 2.5.1b, --alignIntronMin 4 --alignIntronMax 5000 -- alignMatesGapMax 5000). Wig files of uniquely mapped reads were converted in BigWig files with wigToBigWig (v4). StringTie (v2.1.1) were then used to assemble the mapped RNA-Seq reads into transcripts with different parameters depending of the depth sequencing of libraries and type (-m 150 --rf --g 0 -f 0.1 -a 10 -j 2 or -j 4). Trinity script inchworm_transcript_splitter.pl (version 2.8.5) was used to split the transcripts with non-uniform coverage based on the jaccard clip method. Clipped transcripts were extracted with home-made script and clustered with Stringtie and associated bam files to obtain TPM counts. All libraries were concatenated into one gff file without merge to avoid loss of information by fusion of small transcripts into larger ones due to the large number of genes in Z.tritici with overlapping UTRs.
The CupCake package (v10.0.0, https://github.com/Magdoll/cDNA_Cupcake) filtered the isoforms, removing the less expressed and degraded ones using the following tools: collapse_isoforms_by_sam.py, get_abundance_post_collapse.py, filter_by_count.py, filter_away_subset.py. Readthrough transcripts were removed using the previous annotation (MPI, JGI, CURTIN, RRES) with BEDTools intersect with an overlap 100% full CDS (-F 1.0) and same strand (-s)) of at least 2 CDS. Mitochondrial transcripts were filtered out as well. Then, all libraries were processed with chain_samples.py from CupCake and clustered for stringent selection. Splicing junctions obtained by STAR (SJ.out.tab files) from Illumina RNA-Seq libraries were used to filter out isoform transcripts with unsupported junctions. Finally, long-read transcripts fully spanning Transposable Element were removed with BEDTools, giving the final set of transcript evidence.
Assembly: Z.tritici IPO323
Supplementary files format and content: gff format file with isoforms from Iso-Seq data
Supplementary files format and content: gff format file with all transcripts assembles from RNA-Seq data

Submission date

Nov 28, 2022

Last update date

Jul 04, 2023

Contact name

Nicolas Lapalu

Organization name

INRAE

Department

SPE

Lab

BIOGER