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| Status |
Public on Jun 22, 2011 |
| Title |
RNA_21hr_nitrogen_step_down |
| Sample type |
SRA |
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| Source name |
whole filament
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| Organism |
Nostoc sp. PCC 7120 = FACHB-418 |
| Characteristics |
cell type: whole filament
strain: PCC 7120
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| Treatment protocol |
Cells were collected for the 0 hr time point before nitrogen step down, or washed 3 times in BG11 N- and diluted ten fold for diazotrophic growth for 6, 12, or 21 hours. At 6 hours cells are beginning to establish a pattern of proheterocysts, at 12 hours cells are committed to becoming heterocysts, and at 21 hours filaments have fully formed nitrogen-fixing heterocysts. Cells were then collected by cooling 50 ml of culture over 100 g ice and centrifugation at 4,000 x g for 10 minutes at 4°C. Pellets were transferred to a 2 ml tube and spun at 11,500 x g for 2 minutes at 4°C. Excess BG11 was removed and pellets were flash frozen in liquid nitrogen for storage at -80°C.
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| Growth protocol |
For deep sequencing, total RNA was prepared from Anabaena cultures grown in 100 ml flasks in the presence or absence of combined nitrogen as previously described . Briefly, 100 ml of cells were grown to an OD750 of 0.5 in BG11 medium lacking sodium nitrate and containing 5 mM MOPS (pH 8.0) and 2.5 mM ammonium chloride.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated with the Ambion RiboPure RNA isolation kit according to the manufacturer's protocol and total RNA (with ribosomal RNA) was sent for deep sequencing. We chose to include ribosomal RNA to minimize the depletion of coding transcripts by the rRNA removal protocols. In addition, we did not multiplex to avoid bias from using different adapters for different time points, as is required when multiplexing with different data sets. Therefore, a directional RNA sequencing library was prepared from 1 µg of total RNA from each sample (0, 6, 12, 21). The samples were purified using a Qiagen RNeasy MinElute Cleanup Kit, fragmented for 180 seconds using a Covaris S2 set at 10% duty cycle, 5 Intensity, 200 cycles/burst in 120µl Tris-EDTA pH 8.0 and purified again using a Qiagen RNeasy MinElute Cleanup Kit. The fragmented RNA (100 ng) was dephosphorylated using antarctic phosphatase (2 units, 37 °C, 30 min), 5' phosphorylated using T4 polynucleotide kinase (20 units, 37 °C, 60 min) and purified using a Qiagen RNeasy MinElute Cleanup Kit. After this, cDNA library preparation steps were performed as described in the Illumina “Small RNA v1.5 Sample Preparation Guide.” For sequencing, the cDNA library was denatured in 0.1N NaOH and diluted to a final concentration of 9 pM before being loaded onto the Illumina single read flow-cell.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer II |
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| Data processing |
CLC Genomics Workbench 4.0 was used to align reads to the NCBI Nostoc 7120 gbk file for the chromosome and each of seven plasmids with 2 mismatches allowed. Reads with more than 10 total matches were removed from the dataset, as were reads that aligned to the rRNA and tRNA annotated sequences.
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| Submission date |
Jan 14, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
James W Golden |
| E-mail(s) |
[email protected]
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| Phone |
8585349555
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| Organization name |
UCSD
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| Department |
Biological Sciences
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| Lab |
James Golden
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| Street address |
9500 Gilman Dr. MC 0116
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92093 |
| Country |
USA |
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| Platform ID |
GPL11645 |
| Series (1) |
| GSE26633 |
Transcriptional Regulation of Anabaena 7120 response to Nitrogen Deprivation |
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| Relations |
| SRA |
SRX039131 |
| BioSample |
SAMN00198543 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM655512_T21_aligned_CHROMOSOME.bam |
39.0 Mb |
(ftp)(http) |
BAM |
| GSM655512_T21_aligned_pALPHA.bam |
749.0 Kb |
(ftp)(http) |
BAM |
| GSM655512_T21_aligned_pBETA.bam |
584.8 Kb |
(ftp)(http) |
BAM |
| GSM655512_T21_aligned_pDELTA.bam |
100.1 Kb |
(ftp)(http) |
BAM |
| GSM655512_T21_aligned_pEPSILON.bam |
57.0 Kb |
(ftp)(http) |
BAM |
| GSM655512_T21_aligned_pGAMMA.bam |
151.8 Kb |
(ftp)(http) |
BAM |
| GSM655512_T21_aligned_pZETA.bam |
567.9 Kb |
(ftp)(http) |
BAM |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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