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| Status |
Public on Apr 09, 2021 |
| Title |
VT1367_rep_C [Ribo-seq] |
| Sample type |
SRA |
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| Source name |
whole animal
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| Organism |
Caenorhabditis elegans |
| Characteristics |
genotype: maIs105
Stage: mid L4
temperature: 20C
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| Treatment protocol |
Synchronized populations of developing worms were cultured at 20 °C for 45 hrs after feeding. Harvested worms were washed with water three times and incubated at room temperature for 10 min to allow digestion of intestinal bacteria. Worms were then pelleted by centrifuge at 4,500 rcf for 2 min at room temperature and residual water was removed until the total volumes were twice as the worm pellets. The samples were then flashed frozen by liquid nitrogen and stored at -80 °C.
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| Growth protocol |
C.elegans were cultured on nematode growth medium (NGM) and fed with E. coli HB101 unless specified. To obtain populations of synchronized developing worms, gravid adults were collected and washed twice with water. Pellets of centrifuged worms were treated with 5 ml 1N NaOH and 1% (v/v) sodium hypochlorite for 5 min with shaking to obtain embryos, and the embryos were rinsed with M9 buffer three times. The embryos were hatched in 10 ml M9 buffer at 20°C for 16-18 hrs with mild shaking. Hatched L1 larvae were transferred to plates at 30-50 worms per plate and replicate plates were cultured for defined periods; samples of the population were examined by microscopy to confirm the developmental stage at the time of harvest.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Concentrated lysis buffer was add to each frozen sample to final concentration of 20 mM Tris-HCl (pH = 7.4), 150 mM NaCl, 5 mM MgCl2, 0.5X Protease Inhibitor (Sigma, Cat:P2714), 1 mM DTT, 0.1 mg/ml cycloheximide (Millipore, Cat:C4859), 1% (v/v) Triton X100 and 5 U/ml Turbo DNase (Invitrogen, Cat:AM2238), and worm pellets were kept on ice until fully thawed. Suspended worms were transferred to 400 µm silica beads tube (OPS Diagnostic, Cat: PFAW-400-100-04) and lysed in bead beater homogenizer for 3 min at 4 °C. Lysates were then centrifuged at 25,000 rcf for 10 min at 4 °C, and supernatants were collected. To generate monosomes, RNase I (Invitrogen, Cat: AM2294) was added to a final concentration of 0.2 U per µl of harvested worm pellet. The digestion was incubated at room temperature for 40 min with gentle rotation and then quenched by adding SUPERase RNase inhibitor (Invitrogen, Cat: AM2694) at 4 U per RNase I unit. The lysates were then loaded onto 5-40 % (m/v) sucrose gradients prepared with lysis buffer without Triton X100 and centrifuged at 32,000 rpm for 3 hrs at 4 °C in an SW41Ti rotor (Beckman Coulter, Cat:331362). The sucrose gradients were fractionated using BR-188 Density Gradient Fractionation System with 60% (m/v) sucrose as chase solution, and monosome fractions were collected according to OD254 profiles. 3.5 X volumes of QIAzol reagent was added to the gradient fractions containing monosomes, and RNA was extracted and separated by 17.5 % denaturing PAGE. A synthetic RNA oligonucleotide with the length of 30 nt was used as a size marker. The gel was stained by Sybr Gold (Invitrogen, Cat: S11494) at room temperature for 10 min, and a gel slice containing RNA of approximately 30 nt was excised and ground by an RNase-free pellet pestle (Fisher Scientific, Cat: 12-141-364). RNA was extracted from the gel slice by adding 500 µl of 300 mM NaAc (pH = 5.5), 1 mM EDTA, and 0.25% (m/v) SDS and mildly shaking overnight at room temperature. The gel granules were excluded using Spin-X tube filter (Millipore, Cat: CLS8160) and the RNA was then concentrated by ethanol precipitation, dissolved in water, and stored at -80 oC. 5’ phosphorylation and 3’ dephosphorylation were performed with T4 PNK (NEB, Cat: M0201S) following the manufacturer’s instructions, and the products were subjected to phenol/chloroform extraction and ethanol precipitation.
Libraries were constructed using QIAseq miRNA Library Kit (Qiagen, Cat:331505 & 331595) following the manufacturer’s instructions, except that the amplifying PCR was conducted with 9-12 cycles, and sequencing was perform using Illumina NextSeq 500 system.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
WT for U18A
Ribo.raw.reads.VT1367_VT3797.csv
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| Data processing |
Library strategy: Ribo-seq
Adaptor removal and size filtering by Cutadapt/1.9
rRNA/tRNA removal by Bowtie2/2.3.4.3
Genome mapping by Star/2.5.3
RPF p-offset and monosome periodosity calculation by olastid/0.4.8
Gene counting by plastid_cs/0.4.8
Genome_build: WBcel235
Supplementary_files_format_and_content: comma-seperated values of gene counts
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| Submission date |
Apr 08, 2021 |
| Last update date |
Apr 09, 2021 |
| Contact name |
Ye Duan |
| E-mail(s) |
[email protected]
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| Phone |
5088565723
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| Organization name |
Umass Medical School
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| Department |
Program of Molecular Medicine
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| Lab |
Victor Ambros
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| Street address |
373 Plantation Street, Biotech Two, Suite 306
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| City |
Worcester |
| State/province |
MASSACHUSETTS |
| ZIP/Postal code |
01605 |
| Country |
USA |
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| Platform ID |
GPL19757 |
| Series (2) |
| GSE171726 |
Critical contribution of 3’ non-seed base pairing to the in vivo function of the evolutionarily conserved let-7a microRNA [Ribo-seq] |
| GSE171748 |
Critical contribution of 3’ non-seed base pairing to the in vivo function of the evolutionarily conserved let-7a microRNA |
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| Relations |
| BioSample |
SAMN18675300 |
| SRA |
SRX10556684 |
| 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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