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| Status |
Public on Feb 01, 2019 |
| Title |
pmk-1(km25) PA14 2 |
| Sample type |
SRA |
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| Source name |
whole animal
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| Organism |
Caenorhabditis elegans |
| Characteristics |
tissue: whole animal
genotype: pmk-1(km25)
bacterial exposure: PA14
exposure length: 4 hours
age: L4
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| Treatment protocol |
L4 animals were washed to agar plates seeded with non-pathogenic (OP50) or pathogenic (PA14) bacteria for a period of 4 hours at 25 degrees C before RNA was harvested
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| Growth protocol |
Animals were synchronized by bleach prep. Arrested L1s dropped onto agar plates seeded with OP50 and grown at 20 degrees C until the L4 larval stage.
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| Extracted molecule |
total RNA |
| Extraction protocol |
After 3 washes in M9 buffer, TRIzol Reagent (Invitrogen) was added to worm pellets and flash frozen in liquid nitrogen. After an additional round of freeze-thaw, RNA was isolated using the Direct-zol RNA MiniPrep kit (Zymo Research)
Libraries prepped using Kapa mRNA Hyperprep kit. Paired end reads sequenced on Illumina NextSeq500 sequencer, with v2 high-output chemistry.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
D17-178010
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| Data processing |
Base-calling was performed using Illumina's NextSeq control software, RTA 2.4.11
Quality control: Reads were aligned against C. elegans (ws206) using bwa aln/ sampe v. 0.7.16a-r1181 with flags –t 16 –f and mapping rates, fraction of multiply-mapping reads, number of unique 20-mers at the 5’ end of the reads, insert size distributions and fraction of ribosomal RNAs were calculated using dedicated perl scripts and bedtools v. 2.25.0.64.
RNA-Seq mapping and quantitation: Reads were aligned against wbps9/ws258 annotation using STAR v. 2.5.3a with flags -runThreadN 16 --runMode alignReads --outFilterType BySJout --outFilterMultimapNmax 20 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --outFilterMismatchNmax 999 --alignIntronMin 10 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --outSAMtype BAM SortedByCoordinate --quantMode TranscriptomeSAM with --genomeDir pointing to a 75nt-junction wbps9/ws258 STAR suffix array.
Gene expression was quantitated using RSEM v. 1.3.0 with the following flags for all libraries: rsem-calculate-expression --calc-pme --alignments -p 8 --forward-prob 0 against an annotation matching the STAR SA reference. Posterior mean estimates (pme) of counts and estimated RPKM were retrieved.
Genome_build: ws258(wbps9) annotation
Supplementary_files_format_and_content: txt file with, for each gene and each sample, gene length, effective gene length, and posterior mean estimates of read counts (from RSEM).
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| Submission date |
Aug 30, 2018 |
| Last update date |
Feb 01, 2019 |
| Contact name |
Dennis Kim |
| Organization name |
MIT
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| Street address |
31 Ames Street
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
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| Platform ID |
GPL19757 |
| Series (2) |
| GSE119292 |
Transcriptional profiling of C. elegans on pathogenic Pseudomonas aeruginosa |
| GSE119294 |
C. elegans on pathogenic Pseudomonas aeruginosa |
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| Relations |
| BioSample |
SAMN09939123 |
| SRA |
SRX4626818 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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