Methods
Summary
Location and Timeline:
Dissections were performed in Hochberg’s Lab. RNA extractions took place in Garb’s Lab. The library sample preparation and sequencing was carried out at the UMass Lowell Biomolecular Characterization Lab Core Research Facility. The work was conducted between the 09MAR2017 and 16MAR2017.
General Workflow:
1) Two adult females (5th instars) were dissected. Each animal had the following tissues removed and isolated individually into microcentriduge tubes, which were subsequently flash frozen in liquid nitrogen and stored at -80 C.
a. Pygidial glands
b. Hepatopancreas tissue
c. Dorsoventral muscles
2) Tissue was homogenized in trizol, follow Trizol protocol to isolate RNA (https://tools.thermofisher.com/content/sfs/manuals/trizol_reagent.pdf) through step 8.
3) Transfer 100 uL aliquots of aqueous phase containing RNA into separate tubes.
4) Subject those to clean-up using Qiagen RNeasey kit RNA clean up protocol on page 54 of handbook (https://www.qiagen.com/us/resources/resourcedetail?id=14e7cf6e-521a-4cf7-8cbc-bf9f6fa33e24&lang=en)
5) Treat total RNA sample with DNAse using Turbo DNAse kit (https://tools.thermofisher.com/content/sfs/manuals/cms_055740.pdf)
6) The total RNA concentration was measured using the RNA High Sensitivity Assay by fluorometry on the Qubit 3.0 Fluorometer (Product No.: Q32855, Life Technologies).
7) RNA Integrity was measured on an RNA 6000 Nano Bioanalyzer Chip (Product No.: 5067-1511, Agilent Technologies).
8) All samples with large, sharp rRNA peaks by Bioanalyzer analysis, and masses above 250 ng were converted into DNA libraries.
9) 250 ng of total RNA from each sample were converted into DNA libraries by a Illumina Stranded mRNA Library Preparation Custom Protocol using the Illumina LT stranded mRNA Seq Reagents (Catalog No.: 15032612, Illumina Inc.)
10) Library QC was completed on the Agilent Bioanalyzer High Sensitivity DNA chip with High Sensitivity Reagents. Libraries that passed had a mean fragment length of ~260 bp. Libraries were quantitated using the KAPA Biosystems Illumina Library Quantification Kit (Catalog No.: KK4824, Kapa Biosystems).
11) Libraries were normalized to 10 nM concentration and pooled equi-volume with 6 libraries from another mRNA sequencing project for a total plexity of 8-plex, denatured with 0.2N NaOH and loaded into the Illumina NextSeq500 at a concentration of 1.8 pM with a 1% PhiX (Catalog No.: FC-110-3001, Illumina Inc.) spike-in for alignment QC purposes.
12) The libraries were sequenced on the NextSeq 500 Next Generation Sequencer (Illumina Inc.) using a High-Output Sequencing Kit (Catalog No.: FC-404-2004, Illumina Inc.).
13) The raw data from the sequencer are .bcl files and were converted to FASTQ data files using the program bcl2fastq, and FASTQ files were delivered to a shared server for retrieval by the client of the core facility, Esperanza Rivera de Torre.
14) Data analysis was completed by Esperanza Rivera de Torre under the direction of Dr. Jessica Garb.
Protocols
This project has not yet shared any protocols.