|
| Status |
Public on Jun 07, 2018 |
| Title |
28_334_CGTC_8N_L4 |
| Sample type |
SRA |
| |
|
| Source name |
whole worm
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: N2
Stage: L4
bio replicate no: 2
|
| Treatment protocol |
Embryos were isolated from gravid N2 adults by treatment with sodium hypochlorite solution. To obtain synchronized L4 worms, embryos were incubated in M9 media without food at 20 °C for 24h. Hutched synchronized L1 were grown until they reached L4 larval stage.
|
| Growth protocol |
worms were grown at 20°C on enriched NGM with OP50 as food
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Synchronized Embryos or L4 larval worms were washed several times to avoid contamination with bacteria, snap-freezed at liquid nitrogen, and grinded to powder in a liquid nitrogen -prechilled mortar and pestle. High-molecular weight and Low-molecular weight RNA fractions were isolated using miRVana miRNA isolation kit Applied Biosystems (Ambion).
The protocol includes ligation of a total pool of small RNAs first to 3’ adapter pre-adenylated 3’ adapter without ATP, followed by ligation to a 5’-adapter containing 4-nt RNA barcode to allow multiplexing and 8 random nucleotide UMI, reverse transcription and amplification using primers complementary to the 5’ and 3’ adapters, baring Illumina barcodes. 3 size-selection steps are performed, separation of small RNA (20-30b) from longer RNA species and separation, after each one of the two ligations, of small RNA ligated to adapter(s) from free adapter/adapter dimer. All the separations performed on SPRI beads using a proprietary combination of two crowding agents, PEG and Isopropanol. The resulting Libraries were sequenced using 50 bp SR sequencing mode on the HiSeq 2500 platform (Illumina).
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
small RNA
8N identifier
The barcode can be found in each sequence according to the number indicated in the sample name: sequences in samples which include "8N" in their title start with 8 random bases, followed by their barcode, and sequences in samples which include "0N" in their title begin with their barcode.
|
| Data processing |
Library strategy: QsRNA-seq
Filter sequences with irrelevant barcodes
Split relevant sequences by barcode
Remove the linker from each sequence
DEseq package in R (http://www.r-project.org) was used from the data obtained from bowtie to identify differentially expressed miRNAs.
Genome_build: miRBase WBcel235
Supplementary_files_format_and_content: Suppl_Table_3.xlsx (Deseq table)
|
| |
|
| Submission date |
Mar 20, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Ayelet Lamm |
| E-mail(s) |
ayeletla@tx.technion.ac.il
|
| Organization name |
Technion
|
| Department |
Biology
|
| Lab |
Ayelet Lamm
|
| Street address |
Technion st
|
| City |
Haifa |
| ZIP/Postal code |
3200003 |
| Country |
Israel |
| |
|
| Platform ID |
GPL18245 |
| Series (1) |
| GSE96824 |
QsRNA-seq: a method for high-throughput profiling and quantifying small RNAs |
|
| Relations |
| BioSample |
SAMN06621953 |
| SRA |
SRX2657368 |
