|
|
GEO help: Mouse over screen elements for information. |
|
Status |
Public on Sep 28, 2024 |
Title |
Small RNA sequencing of High Worms biological replicate 2 |
Sample type |
SRA |
|
|
Source name |
whole multiple worms
|
Organism |
Caenorhabditis elegans |
Characteristics |
strain: BCN1050 unc-119(ed3) III; crgIs1002[pdaf-21::mCherry::unc-54 3'UTR; unc-119(+)] strain background: N2 developmental stage: gravid adult condition: grown at high temperature
|
Growth protocol |
To prepare the ‘High’ and ‘Low’ daf-21p::mCHERRY populations for RNA extraction, P0 worms kept at 16 and 25 ºC were transferred to 20 ºC as L4s (12 worms/plate, 3 replicates per treatment). Four and a half days later, when much of the F1 progeny was gravid, the worms were bleached, washed 3x with M9 and the F2 larvae let to hatch o/n in M9 at 20 ºC. The F2 L1 larvae were washed and plated at a concentration of 1000 worms/plate on standard NGM plates seeded with OP50 bacteria. After 72 hours of growth at 20 ºC, the gravid worms were gently washed of the plates and RNA was extracted.
|
Extracted molecule |
total RNA |
Extraction protocol |
RNA was extracted using TRIzol® extraction protocol (Portman, D.S. (2006). Profiling C. elegans gene expression with DNA microarrays. WormBook : the online review of C elegans biology, 1-11.) 1 microgram of total RNA was treated with Antarctic phosphatase (ref. M0289S, NEB) and subsequently with T4 PNK (3' phosphatase minus) (ref. M0236S, NEB) in order to make the 5’ and 3’ ends of the RNA available for adapter ligation. Samples were further processed using the TruSeq small RNA Sample Prep Kit (ref. RS-200-0012, Illumina) according to the manufacturer's protocol. Briefly, 3’ adapters and subsequently 5’ adapters were ligated to the RNA. cDNA was synthesized using reverse transcriptase (SuperScript II, ref. 18064-014, Invitrogen) and a specific primer (RNA RT Primer) complementary to the 3’ RNA adapter. cDNA was further amplified by PCR using indexed adapters supplied in the kit. Finally, libraries were size selected using 6% Novex® TBE Gels (ref. EC6265BOX, Life Technologies). Fragments with insert sizes of 18 to 36 bp were cut from the gel, and DNA was precipitated and eluted in 10 µl EB. Final libraries were analyzed using Agilent DNA 1000 chip to estimate the quantity and check size distribution, and were then quantified by qPCR using the KAPA Library Quantification Kit (ref. KK4835, KapaBiosystems) prior to amplification with Illumina’s cBot. Libraries were pooled and loaded at a concentration of 10 pM onto the flowcell, and were sequenced 1 x 50 on Illumina’s HiSeq 2000.
|
|
|
Library strategy |
miRNA-Seq |
Library source |
transcriptomic |
Library selection |
size fractionation |
Instrument model |
Illumina HiSeq 2000 |
|
|
Description |
High
|
Data processing |
Adaptor trimming using cutadapt v1.7.1 -a TGGAATTCTCGGGTGCCAAGGAACTCCAGTCAC -O 6 -m 15 Sequence mapping using bowtie v1.1.1 -a --best --strata -v 0 Assigning reads to genes using featureCounts v1.4.6 -s 2 -t exon -g gene_name -M -O Counts normalization by total mapped small RNAs using DESeq2 v1.8.1. Genome_build: C. elegans genome assembly WS235 from WormBase and annotation from Ensembl release 80 with the vector transgene as an additional chromosome Supplementary_files_format_and_content: Matrix of scaled number of molecules targeting each gene. The individual files representing 22G, 21U, and 26G represent the normalized counts of a specific small RNA class consiting of (22, 21 or 26) nucleotides and starting with the letter (G or U).
|
|
|
Submission date |
Oct 29, 2015 |
Last update date |
Sep 28, 2024 |
Contact name |
Eduard Casas |
E-mail(s) |
eduard.casasmasnou@colorado.edu
|
Organization name |
CU Boulder
|
Department |
MCDB
|
Lab |
Brumbaugh Lab
|
Street address |
Porter Biosciences, CU Campus
|
City |
Boulder |
State/province |
Colorado |
ZIP/Postal code |
80302 |
Country |
USA |
|
|
Platform ID |
GPL13657 |
Series (1) |
GSE74496 |
Trans-generatioanl epigenetic inheritance and resetting of an environmentally-triggered change in gene expression in C. elegans |
|
Relations |
BioSample |
SAMN04226127 |
SRA |
SRX1399066 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
|
|
|
|
|