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Sample GSM948508

Query DataSets for GSM948508

Status

Public on Sep 21, 2012

Title

Uninfected polysomal Set 3

Sample type

SRA

Source name

midgut

Organism

Anopheles gambiae

Characteristics

strain: G3
time post bloodmeal: 22-26 hrs
gender: female
infection status: uninfected control

Treatment protocol

Gametocytes were harvested and used immediately in bloodfeeding of mosquitoes. Female adult An. gambiae (G3 strain) mosquitoes were maintained on 10% sucrose at 27ºC/80% humidity. Prior to bloodfeeding, the mosquitoes (4-6 days post-eclosion) were maintained overnight on water only. Bloodfeeding was carried out using a Hemotek 5W1 system and collagen Hemotek feeding membranes (Discovery Workshops, Lancashire, England). Mosquitoes were allowed to feed for 15 minutes. This resulted in bloodfeeding of approximately 80-90% of available Anopheles gambiae females. A control group of mosquitoes were fed on uninfected blood and serum by essentially the same method.

Extracted molecule

total RNA

Extraction protocol

Two hundred female mosquitoes were dissected at 22-26 hours post blood meal to obtain midguts. The samples were flash frozen in liquid nitrogen and stored at -80ºC until processed. Frozen samples were ground using a mortar and pestle, homogenized in 1 ml lysis buffer (15 mM Tris-HCl, pH 8.0, 300 mM NaCl, 5 mM MgCl2, 0.5 mM DTT, 0.1 mg/ml cycloheximide, 1 mg/ml heparin, 1% triton X-100, 0.2 U/ml RNase inhibitor). The extracts were spun 15 minutes at 14,000 rpm at 4ºC to remove cell debris and nuclei. An aliquot of the supernatant was saved for analysis of total cellular mRNA. The rest of the supernatant was directly applied to the top of a 10-60% linear sucrose gradient that was prepared as described by Arava, containing 20 mM Tris–HCl (pH 8), 140 mM KCl, 5 mM MgCl2, 0.5 mM DTT, 0.1 mg/ml cycloheximide, and 0.5 mg/ml heparin. Centrifugation was carried out using an SW41-Ti rotor at 116,000×g for 3 hours at 4ºC. Following spinning, gradient fractions were collected immediately using a Density Gradient Fractionation System (Brandel, Gaithersburg, MD) with simultaneous recording of absorbance profiles at 254 nm. Following fractionation, non-polysomal aliquots were pooled together into one sample, and polysomal aliquots were pooled into a second sample. The fraction between was discarded to ensure good separation. RNA samples from three independent infection experiments were collected. For each set, we had six RNA samples: polysomal and nonpolysomal RNAs of the infected mosquitoes, polysomal and nonpolysomal RNAs of the uninfected controls, and total cellular mRNAs of the infected and uninfected mosquitoes. A fourth set of samples were maintained as individual fractions for qRT-PCR analysis of specific transcripts. RNA was extracted from the fractionated and unfractionated samples using Trizol reagent (Invitrogen, Carlsbad, CA). Sample preparation for Illumina-based mRNA sequencing was conducted based upon the manufacturer’s protocols “mRNA Sequencing Sample Preparation Guide” and “Preparing Samples for Multiplexed Paired-End Sequencing” (Part# 1005361Rev B, and Part# 1004898Rev D, Illumina, Inc., San Diego, CA). For each set, six cDNA libraries derived from the six RNA samples were uniquely tagged with index sequences at the PCR stage of sample preparation. Six libraries were pooled together and sequenced in a single lane of a flow cell. 76-cycle single read Illumina sequencing was conducted at the Virginia Bioinformatics Institute (VBI, Blacksburg, VA).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina Genome Analyzer IIx

Data processing

Illumina Casava1.6 software used for basecalling and demultiplexing; adaptor removal using a perl script. These steps conducted by Virginia Bioinformatics Institute.
Convert Fastq to Fasta using Tu lab program "fastq_to_fasta" (Jake Tu, Virginia Tech)
Format Fasta using the program "formatdb"
BLAST: Blastn against An. gambiae database using the following conditions: -p blastn -a 12 -m 8 -v 1000000 -b 1000000 -e 1e-20 -m 8 -d
Tally results: Count number of hits for each query using Tu lab program "Count_query.pl" and merge results into a single table with the Tu lab program "mergeHitCount_adv.pl" (Jake Tu, Virginia Tech)
Genome_build: An. gambiae P3.4 CDNA ALL from Vectorbase
Supplementary_files_format_and_content: Excel file includes RPM (Reads Per Million) values for each sample and the calculated relative polysome loading for each mRNA species.

Submission date

Jun 13, 2012

Last update date

May 15, 2019

Contact name

Jinsong Zhu

E-mail(s)

zhujin@vt.edu

Phone

540-231-3841

Organization name

Virginia Tech