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| Status |
Public on Mar 21, 2024 |
| Title |
Day6 Wild-type(N2) feed with E. coli OP50, replicate 3 |
| Sample type |
SRA |
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| Source name |
whole body
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| Organism |
Caenorhabditis elegans |
| Characteristics |
tissue: whole body
strain: Wild-type(N2)
age: day6
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| Extracted molecule |
nuclear RNA |
| Extraction protocol |
Worms were washed three times with PBS and collected in a 1.5 mL tube. We then added 100 μl of homogenization buffer {10.1126/science.abk2432} and grind the worms with a pestle motor for 30 seconds on ice. To minimize nuclei adhesion on the surface, all the pestles, tubes and filters were pre-coated with a homogenization buffer or 1x PBS. 900 μl of homogenization buffer was added to wash the pestle, and the total 1mL homogenized sample was transferred into a 1mL Dounce tissue grinder (Wheaton 357538). The grinder was autoclaved overnight in a 220℃ oven to deactivate ribonuclease. After placing the grinder on ice, 20 strokes were applied using a loose pestle, followed by another 20 strokes using a tight pestle, while avoiding generating foams. The 1000μl samples were filtered through a cell strainer (35 μm) and then through a Flowmi cell strainer (BelArt, H13680-0040) into a new 1.5 mL tube. The tubes were centrifuged for 10 minutes at 1000 g at 4°C, and the supernatant was removed while not disturbing the pellet, which was hardly visible. The pellet was resuspended with 500 μl 1x PBS with 0.5% BSA and RNAase inhibitor and filtered with a Flowmi cell strainer (BelArt, H13680-0040) again into a 5 mL flow cytometer tube. A total of 20 μl was transferred into another flow cytometer tube and diluted with 180 μl 1x PBS with 0.5% BSA and RNAase inhibitor as an unstained control. Hoechst (Invitrogen 33342) was used to stain the nuclei in 1:1000 working concentration. We coated the 1.5 mL sorting collection tube with 1x PBS with 0.5% BSA and RNAase inhibitor and ~300,000 nuclei were sorted with Hoechst 33342 positive gating, which indicates DNA content, and forward scatter area (FSC-A) gating, representing the particle size above threshold. The intestinal nuclei, which are polypoid (32N), usually form an obvious cluster separating from the other 2N somatic nuclei, and these were also included in our collection. The collection tube was centrifuged at 800g for 8 minutes at 4℃ and the sheath buffer supernatant was carefully removed before resuspending the sorted nuclei with 40-50 μl 1x PBS with 0.5% BSA and RNAase inhibitor. The concentration and morphology of the nuclei were checked under a microscope to ensure high-quality nuclei isolation. If the results were desirable, we proceeded to generate gel emulsion with 10X Chromium Controller. For sorting the nuclei, we used either DB LSR II or Sony MA800 sorter. To obtain the best nuclei morphology and the least RNA degradation, it is recommended to minimize the sorting time. Two researchers could simultaneously set up the sorter and perform nuclei processing.
Library preparation was carried out using the published 10X Chromium Single Cell 3’ v2/v3 Solution protocol (Single/dual index). The resulting libraries were sequenced on the NextSeq550 or NovaSeq6000 or NextSeq 2000 platforms, with a depth ranging from 6,306 to 29,862 reads/cell, with the recommended cycle numbers: 26 cycles for Read 1, 8 cycles for i7 index, and 98 cycles for Read 2.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
NextSeq 2000 |
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| Description |
10x Genomics
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| Data processing |
We used Cell ranger (6.0, 10x Genomics) to align Raw base call (BCL) sequence files or FASTQ files to the C. elegans genome (WS282, Wormbase), and generated feature-barcode matrices. Doublets were removed based on the recommended ratio provided by the 10X Genomics Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 user guide (CG000204 Rev D), with a score calculated by Doubletfinder (Github/chris-mcginnis-ucsf). The feature-barcode matrices for each sample were constructed into a Seurat (R package, 4.0.5) object for downstream analysis. Cells were filtered by requiring a minimum of 100 genes to be expressed, and genes were filtered by being expressed in at least 3 cells. Integration of samples was performed with Seurat canonical correlation analysis (CCA) method to remove the batch effects. We tested CCA, reciprocal principal component analysis (PCA) and Harmony (0.1) for integration and chose the method with the best performance. The top 2,000 variable features were used for integration anchor identification. UMAP and tSNE dimension reduction were performed with the first 50 dimensions from PCA. Clustering was performed at multiple Leiden resolutions.
Assembly: WS282
Supplementary files format and content: Tab-separated values files and matrix files
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| Submission date |
Jan 22, 2024 |
| Last update date |
Mar 21, 2024 |
| Contact name |
Shihong Max Gao |
| E-mail(s) |
gaos2@hhmi.org
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| Organization name |
Janelia Research Campus
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| Department |
4DCP
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| Lab |
Meng Wang
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| Street address |
19700 Helix Dr
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| City |
Ashburn |
| ZIP/Postal code |
20147 |
| Country |
USA |
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| Platform ID |
GPL32326 |
| Series (1) |
| GSE229022 |
Single nuclei gene expression profile for adult C. elegans and several longevity mutants |
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| Relations |
| BioSample |
SAMN39550234 |
| SRA |
SRX23352754 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8029694_N2D6R2_barcodes.tsv.gz |
37.9 Kb |
(ftp)(http) |
TSV |
| GSM8029694_N2D6R2_features.tsv.gz |
380.9 Kb |
(ftp)(http) |
TSV |
| GSM8029694_N2D6R2_matrix.mtx.gz |
20.3 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
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