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| Status |
Public on Nov 18, 2023 |
| Title |
SFAV1_reRNA_5dpi_hemolymph_sRNAseq |
| Sample type |
SRA |
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| Source name |
Hemolymph
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| Organism |
Spodoptera frugiperda |
| Characteristics |
tissue: Hemolymph
treatment: wild-type ascovirus
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| Extracted molecule |
total RNA |
| Extraction protocol |
The cell pellets were resuspended in 98% formamide and 10 mM EDTA and lysed at 70 °C for 10 min. The total RNA were then extracted from the lysate using Zymo Quick-RNA Miniprep Kit (Zymo Research) and used for small-RNA sequencing library preparation.
Following on-column DNAse treatment, total RNA was eluted using water. ~200 ng of total RNA was used for rRNA depletion reactions using NEBNext rRNA kit (bacteria) following manufacturer protocols. For eukaryotic samples, custom probes designed using NEBNext Custom rRNA depletion tool were used. The rRNA depleted RNA was purified using a Zymo-Spin IIC column, treated with T4 PNK using T4 PNK buffer (NEB) at 37 C for 30 minutes before spiking in ATP and further incubating. Further end treatment was performed using mRNA Decapping Enzyme (NEB) pre-mixed with its buffer directly into the PNK reaction and incubating at 37 C for 30 minutes. End-repaired RNA was purified using a Zymo-Spin IIC column, and used for Collibri small-RNA-seq library preparation reactions following manufacturer protocols (INVITROGEN). The resulting NGS adapter ligated cDNA was then amplified with KAPA HiFi HotStart ReadyMix using NGS indexing primers for 25 cycles (ROCHE). Amplified DNA fragments between 150~500nt were size selected by gel extraction purification following gel-electrophoresis in a 4% SYBR-GOLD E-GEL. Before loading into the sequencer, library concentrations were quantified using KAPA-QUANT qPCR kit (ROCHE).
Bulk RNA was subjected to rRNA depletion with NEBNext rRNA Kit for either bacteria or eukaryotes, depending on the biosample. The final library was prepared with the Collibri small-RNA-seq kit (INVITROGEN) according to manufacturer instructions.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina MiSeq |
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| Data processing |
NGS reads were merged using BBMerge using a normal merge rate and then mapped onto the TnpB containing locus using Geneious mapper at Low Sensitivity to identify reRNA boundaries (Geneious 2023.0.1 (https://www.geneious.com)).
Assembly: For each sample, a custom reference fasta was used for alignment, as described for each processed file below. Reference fastas are supplied under supplementary files.
Supplementary files format and content: MA_4_ISXfa1_IS607_Figure3A.bw was generated by aligning raw data from Figure_3A_IS607_sRNA to ISXfa1_IS607_Contig_Figure3A.fasta, then converted to BigWig Format with DeepTools (v3.5.1).
Supplementary files format and content: SFAV1_5dpi_sRNA.bw was generated by aligning raw data from Figure_5A_SFAV1_sRNA to AM398843_SFAV_IN_VIVO_2And5dpi_FIGURE_5_and_S5.fasta, then converted to BigWig Format with DeepTools (v3.5.1).
Supplementary files format and content: SFAV_2dpi.bw was generated by aligning raw data from Figure_S5A_SFAV1_sRNA to AM398843_SFAV_IN_VIVO_2And5dpi_FIGURE_5_and_S5.fasta, then converted to BigWig Format with DeepTools (v3.5.1).
Supplementary files format and content: sfav_baculo.bw was generated by aligning raw data from Figure_S5B_SFAV1_sRNA to SfAV-1_SF9_contig_FIGURE_S5.fasta, then converted to BigWig Format with DeepTools (v3.5.1).
Supplementary files format and content: hagv_sRNA.bw was generated by aligning raw data from Figure_S5B_HAGV1_sRNA to HAGv-1_SF9_contig_FIGURE_S5.fasta, then converted to BigWig Format with DeepTools (v3.5.1).
Supplementary files format and content: hvav.bw was generated by aligning raw data from Figure_S5B_HVAV1_sRNA to HvAV-1_SF9_contig_FIGURE_S5.fasta, then converted to BigWig Format with DeepTools (v3.5.1).
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| Submission date |
Oct 24, 2023 |
| Last update date |
Nov 18, 2023 |
| Contact name |
Marena Isabelle Trinidad |
| E-mail(s) |
marenatrinidad@berkeley.edu
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| Organization name |
Innovative Genomics Institute
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| Street address |
2151 Berkeley Way
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| City |
Berkeley |
| State/province |
CA |
| ZIP/Postal code |
94704 |
| Country |
USA |
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| Platform ID |
GPL33862 |
| Series (1) |
| GSE246134 |
Eukaryotic RNA-guided endonucleases evolved from a unique clade of bacterial enzymes |
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| Relations |
| BioSample |
SAMN37950001 |
| SRA |
SRX22203586 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7856895_SFAV1_5dpi_sRNA.bw |
42.6 Kb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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