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| Status |
Public on Dec 01, 2012 |
| Title |
Mp708 Treated Midgut Replicate D |
| Sample type |
SRA |
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| Source name |
Midgut tissue from last instar larvae fed on resistant Mp708 maize
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| Organism |
Spodoptera frugiperda |
| Characteristics |
insect strain: Corn Strain
tissue: midgut
Stage: last instar larvae
treatment: reared on susceptible Mp708 maize
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| Treatment protocol |
Larvae were reared in an environmental chamber at 27°C, 14:10 (light:dark) photoperiod, and 70% relative humidity. Midguts were dissected from feeding larvae 2 d after molting to the last instar with masses between 300 and 400 mg. Dissections were done with cold anesthetized larvae submerged in Bombyx saline. After removing Malpighian tubules, foregut anterior to the stomodial valve, hindgut and food bolus, the midgut was transferred from the body cavity, rinsed well with cold saline, and preserved in RNAlater®.
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| Growth protocol |
Beginning as neonates, fall armyworm larvae used in the mRNA-seq experiment were reared on yellow-green midwhorl foliage from resistant Mp708 maize or susceptible Tx601 maize. Old foliage and frass were removed every other day and replaced with fresh foliage.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Isolation of total RNA was performed separately for each midgut from an individual larva. Nearly all RNAlater® was removed after centrifugation. Several 2.3 mm diameter stainless steel beads were added to the concial tube containing a midgut and everything was frozen in liquid nitrogen. Tubes were transferred to adapter blocks prechilled at -20°C and tissue was first disrupted (20 Hz, 45 sec) and then homogenized in TRIzol® reagent (20 Hz, 3 min) on a mixer milll. Isolation of RNA from the homogenate followed the TRIzol manufacturer’s protocol (Invitrogen), except for additional acid phenol:chloroform:isoamyl alcohol (25:24:1, v:v:v) and chloroform phase separation steps prior to RNA precipitation to reduce DNA contamination. Agilent Bioanalysis (RNA Nano Chip) indicated that the isolated total RNA had no detectable DNA contamination and RNA integrity numbers ranged from 8.3 to 9.7. Equal amounts (3 µg) of total RNA from an individual MG were randomly pooled into three replicates per treatment (i.e., Mp708 or Tx601) such that each treatment replicate derived from 12-13 MGs. Each pool of total RNA was separately enriched for poly(A+) RNA using the MicroPoly(A)Purist™ Kit (Applied Biosystems/Ambion) according to the manufacturer’s instructions. Enrichment involved two rounds of oligo(dT) selection with a fresh spin column used in each round. The resulting poly(A+) RNA showed little degradation of the mRNA with a broad peak ranging from 200 nt to 5000 nt centered at about 1880 nt (Agilent Bioanalyzer RNA Pico Chip). Estimated rRNA contamination ranged from 3.2% to 6.1%.
The Penn State Genomics Core Facility (University Park, PA) prepared transcriptome libraries and performed sequencing. Libraries were separately prepared from each of the six poly(A+) RNA samples using the strand-specific SOLiD™ Total RNA-Seq Kit (Applied Biosystems) according to the manufacturer’s protocol. Briefly, samples were fragmented with RNAse III and purified. Fragmented RNA (100 ng) was ligated overnight to adaptors and subsequently reverse transcribed. Purified cDNA was size selected and then amplified using barcoded primers. The resulting material was purified and assessed for proper amplification using an Agilent Bioanalyzer DNA 1000 chip (Agilent Technologies).
Barcoded libraries were combined in equimolar quantities and prepared for sequencing according to the “Applied Biosystems SOLiD 3 Plus System Templated Bead Preparation Guide.” Barcode sequencing and fragment library sequencing (50 bp) was performed on a full plate run of the SOLiD 3 Plus System according to the “Applied Biosystems SOLiD 3 Plus System Instrument Operation Guide.”
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
AB SOLiD System 3.0 |
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| Data processing |
Filtering and Trimming: SOLiD sequence reads were filtered using NextGENe® version 2.0 (SoftGenetics, State College, PA). Reads accepted had a median score threshold ≥12, contained ≥25 bases and contained one or more bases with a score ≥14.
Reference preparation: The 138911 Sanger ESTs in SPODOBASE (http://bioweb.ensam.inra.fr/spodobase) were assembled into a reference transcriptome using SeqMan Pro version 8.0.2 (DNASTAR Lasergene®, Madison, WI) and default parameters for Sanger sequences as recommended by the manufacturer. Preassembly involved a medium end trim, a vector scan and contaminant sequence screen that used a minimum of 5 mismatches; vector and contaminant sequence catalogs were those provided by the software. Assembly parameters were match size of 25, minimum match of 85%, match spacing of 150, minimum sequence length of 100, gap penalty of zero, gap length of 0.7 and maximum mismatch end bases of 15. Consensus calling used primary trace evidence of 50%, a majority of 75%, and quality weights of nine before and 400 after. Calling conflicts and single nucleotide polymorphisms (SNPs) used a minimum coverage of four, minimum inconsistency of 25% and two bases, and no pairing was specified. The assembled reference contained 7869 contigs (7,049,947 nt) that more correctly should be considered gene models.
Mapping: The reads from each library representing a replicate within a maize inbred treatment were mapped separately to the reference transcriptome using the Bowtie-like algorithm in NextGENe® with the requirement that 85% of 12 or more nucleotides comprising a read must match the reference. A read was allowed to map only once (i.e., no ambiguous mapping). The number of mapped reads per gene in each library, representing a replicate within a maize inbred treatment, were summed by NextGENe® as read counts per gene. These data representing transcript abundance were subsequently used in differential expression analyses.
Differential expression: The R software package (v2.15.0) was used to confirm the overall data quality (frequency distribution histogram plots, hexagonal binning plots with R hexbin package v1.26.0, MA plots with R edgeR package v2.6.7, complete linkage cluster dendrogram plots, frequency histograms of Fisher’s exact test P-values with R SAGEnhaft package v1.22.0), filter low expressing genes, and normalize the read counts. Low expressing genes were filtered by summing raw read counts per gene across replicate libraries within a maize inbred treatment; a gene was deleted when this sum for either treatment was less than 20. The filtered read count data were normalized using the trimmed mean of M values (TMM) method as follows; a scaling factor was computed for each replicate library within a maize inbred treatment via the normFact routine in edgeR using filtered read count data and the A-replicate of the Tx601 treatment as a reference. Filtered counts for each gene within a mapped library were divided by the product of the scaling factor multiplied by the library size. Normalized read counts were used to perform two-tailed paired t-tests for each gene. Estimated P-values from each t-test and log2 fold change values for each gene were used to create a volcano plot. False discovery rate for differentially expressed genes was set at an experiment-wide rate of 5% using the Q-value method (R qvalue package v1.18.0).
Orthologous functional annotation: Functional annotation of contigs in the reference used an approach that first obtained a full-length lepidopteran ortholog (BLASTx) against predicted proteins from the whole genome sequence of Bombyx mori [SilkDB version 2.0). These protein orthologs were in turn used to search (BLASTp) and obtain functional orthologs from Drosophila melanogaster (predicted proteins in Flybase release FB2012_04). Functional annotations of genes with significant differential expression were compared to those of all midgut genes in the filtered read count dataset using GO analysis (DAVID v6.7).
Genome_build: Not applicable
Supplementary_files_format_and_content: A tab delimited text file (Filtrd&NormalizdReadCountsSeqsAnnot.txt) contains processed data used to form conclusions in the published manuscript. This file contains information about the reference contigs to which sequence reads were mapped (e.g., contig assembly data, contig sequence, best-hit BLAST annotation results) is given in the data table in addition to the final processed mRNA-seq data (e.g., raw and normalized read counts, t-test P-values, fold differences). The file only contains data for contigs that passed the low expressing gene filter.
Supplementary_files_format_and_content: The matched.csfasta contain processed reads consisting of filtered and trimmed sequence reads that mapped to the reference transcriptome.
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| Submission date |
Nov 30, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Howard W Fescemyer |
| E-mail(s) |
hif1@psu.edu
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| Phone |
814-863-9269
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| Organization name |
The Pennsylvania State University
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| Department |
Biology
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| Street address |
208 Mueller Lab
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| City |
University Park |
| State/province |
PA |
| ZIP/Postal code |
16802 |
| Country |
USA |
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| Platform ID |
GPL16344 |
| Series (1) |
| GSE42659 |
Insect larvae fed on resistant Zea mays (corn) expressing maize insect resistant 1 cysteine protease (Mir1-CP) compensate for Mir1-CP degradation of their peritrophic membrane by altering expression of midgut transcripts as revealed through mRNA-seq |
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| Relations |
| SRA |
SRX207931 |
| BioSample |
SAMN01821847 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1047535_MP_D_solid0565_20100811_Fescemyer_TXA_MPE_6trans_50bpBC_F3_converted_matched.txt.gz |
1.0 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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