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| Status |
Public on Mar 13, 2014 |
| Title |
F. cylindrus RNA sequencing |
| Sample type |
SRA |
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| Source name |
Whole cells
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| Organism |
Fragilariopsis cylindrus |
| Characteristics |
strain: CCMP1102
library strategy: RNA sequencing
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| Growth protocol |
Marine algae (CCMP strains) were grown at the Provasoli-Guillard National Center for Marine Algae and Microbiota. A live culture of Cyanidioschyzon merolae (NIES-1332) was obtained from the Microbial Culture Collection at the National Institute for Environmental Studies, Japan. Cryptococcus neoformans dnmt5Δ (D632) and control (WT47; sxi1Δ) strains were obtained from the Fungal Genetic Stock Center. Strains D632 and WT47 were grown overnight at 23 °C in the dark to saturation in liquid Difco YM Broth (Becton, Dickinson and Company) and 10 μl was patched on autoclaved cellophane overlaid on Difco YM Agar (Becton, Dickinson and Company) and grown for 3 days at 23 °C in the dark. The cells and cellophane were placed in tubes with 0.5 mm Zirconia-Silica beads (BioSpec) and frozen in liquid nitrogen. The tubes were bead-beaten 4 × 1 min. with replacement in liquid nitrogen in between and pulverized material was stored at -80 °C.
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| Extracted molecule |
total RNA |
| Extraction protocol |
The Provasoli-Guillard National Center for Marine Algae and Microbiota prepared genomic DNA and total RNA from marine algae (CCMP strains) and also provided frozen cells. Live Cyanidioschyzon merolae cells were collected by centrifugation and genomic DNA was isolated with a DNeasy Plant Mini Kit (QIAGEN). Purified wild-type Cryptococcus neoformans var. grubii H99 DNA (208821D) and Leishmania major strain Seidman DNA (PRA-309D) were obtained from ATCC. Pulverized Cryptococcus neoformans material from strains D632 and WT47 was partially thawed and genomic DNA was extracted with a DNeasy Plant Mini Kit by starting at the step with addition of AP1 and RNase, and total RNA was extracted with TRIzol (Life Technologies) using Phase Lock Gel Heavy 1.5 ml tubes (5 PRIME).
Assaying cytosine methylation by bisulfite sequencing of genomic DNA was performed as described previously (Ibarra et al., Science 2012). Briefly, genomic DNA was quantified with Qubit dsDNA Assay Kits (Life Technologies) and 50 to 250 ng were sonicated, end-repaired and ligated to methylated adapters. We performed 3 PCR reactions and combined the products before sequencing.
For RNA sequencing, total RNA was digested with RNase-free DNase I (QIAGEN) and concentrated with a RNeasy MinElute Cleanup Kit (QIAGEN). Purified total RNA was quantified by a Qubit RNA Assay Kit (Life Technologies), and between 67 and 125 ng was used to prepare a strand-specific library with the Encore Complete RNA-Seq Library System I (NuGEN).
Assaying nucleosomes by micrococcal nuclease (MNase) sequencing was performed as described previously (Teves and Henikoff, Methods Mol Biol 2012) with modification. Briefly, frozen Ostreococcus lucimarinus or Micromonas pusilla cells from 5 L of culture were thawed on ice and resuspended in 500 μl of cold 1× TM2 (10 mM Tris-HCl, pH 8, 2 mM MgCl2, supplemented with 1:200 EDTA-free plant protease inhibitors (Sigma)) per 50 mg wet-pellet mass by pipetting with a 5-ml pipet up and down 100 times. The slurry was filtered through a 40 μm-pore cell strainer (BD Falcon) by gravity. Triplicate reactions from the same cells were performed: five hundred μl of the cell suspension per reaction were collected by gentle, refrigerated centrifugation and washed once with 1 ml of 1× TM2. Cells were centrifuged again, resuspended in 200 μl of 1× TM2, and warmed to 37 °C for 5 min. Two hundred μl of 1× buffer (50 mM Tris-Hcl, pH 7.9, 5 mM CaCl2), supplemented with 100 μg ml-1 BSA and 125 gel units ml-1 for O. lucimarinus or 250 gel units ml-1 for M. pusilla (~12.5 and 25 Kunitz units ml-1, respectively, which we previously determined to yield mostly mononucleosomes) of micrococcal nuclease (NEB) were pre-heated to 37 °C and added to the 200 μl of resuspended cells, and the 400-μl mixture was further incubated at 37 °C for 10 min. with occasional vortexing. Reactions were stopped with 5 mM final EGTA. Then 100 mM final NaCl, 0.625% final SDS, 20 μg DNase-free RNase A and 50 μg proteinase K were added, and the mixture was incubated at 75 °C for 10 min. Genomic DNA was purified first by extraction with buffer-saturated phenol-chloroform-isoamyl alcohol using Phase Lock Gel Heavy 1.5 ml tubes, and then with 2 volumes of Agencourt AMPure XP beads (Beckman Coulter). For sequencing in vivo nucleosome positions, 66.7 ng from each of the triplicate reactions was combined (200 ng total) and made into a library without PCR by using the Encore Rapid Library System (NuGEN).
For in vitro nucleosome position analyses, we first generated an unmethylated equivalent of O. lucimarinus genomic DNA in vitro by isothermal multiple strand displacement amplification, using either Bst 2.0 (NEB) or an illustra Ready-To-Go GenomiPhi v3 Kit (GE Healthcare). For Bst 2.0, 15 replicate 50-μl reactions containing 10 ng O. lucimarinus genomic DNA each in 1× NEB Isothermal Amplification Buffer with 12.5 μM random hexamers, 400 μM dNTPs (with the deoxycytidine triphosphate unmethylated) and 8 units of Bst 2.0 DNA polymerase (New England Biolabs) were incubated at 50 °C for 8 hr. The DNA was amplified more than 20-fold in these reactions, so that approximately 95% of the amplified DNA molecules contain entirely unmethylated cytosines. The resulting amplified DNA and natively methylated genomic DNA were incubated separately at 75 °C for 10 min at a concentration of 4 μg ml-1 each with 0.62% SDS, 38 ng ml-1 DNase-free RNase A and 100 ng ml-1 proteinase K. DNA was purified by extracting twice with buffer saturated phenol-chloroform-isoamyl alcohol using Phase Lock Gel Heavy 1.5 ml tubes. The DNA was further purified and concentrated using 0.7 volumes of Agencourt AMPure XP beads. The purified natively methylated O. lucimarinus genomic DNA or unmethylated equivalent was assembled with purified recombinant human histones into nucleosomes by salt dilution using an EpiMark Nucleosome Assembly Kit (New England Biolabs) at a 1:2:1 molar ratio of DNA:histone H2A/H2B dimer:histone H3.1/H4 tetramer (assuming 1 nucleosome per 184 bp DNA for O. lucimarinus). The assembly began at 640 nM of 184-bp DNA in 2 M NaCl and was completed when the final concentration of 184-bp DNA was 80 nM and NaCl was 250 mM. The assembly was heated to 37 °C for 5 min and then mixed with an equal volume (80 μl) of prewarmed 1× buffer (50 mM Tris-Hcl, pH 7.9, 5 mM CaCl2), supplemented with 100 μg ml-1 BSA and 250 gel units ml-1 micrococcal nuclease (25 Kunitz units ml-1, which we previously determined to yield mostly mononucleosomes for assembly on either methylated or unmethylated DNA) and incubated at 37 °C for 10 min. Reactions were stopped with 5 mM final EGTA. Then 100 mM final NaCl, 0.625% final SDS and 20 μg proteinase K were added, and the mixture was incubated at 75 °C for 10 min. Nucleosomal fragments were purified first by extraction with buffer-saturated phenol-chloroform-isoamyl alcohol using Phase Lock Gel Heavy 1.5 ml tubes, then with 2 volumes of Agencourt AMPure XP beads, and 90 ng of each sample was made into a library without PCR by using the Encore Rapid Library System.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
genome build: JGI build v1.0
Analyses were performed with Perl scripts from dzlab-tools (http://dzlab.pmb.berkeley.edu/tools/) and custom scripts written in AWK and R (http://www.r-project.org/). bs-sequel from dzlab-tools was used to map bisulfite sequencing reads with Bowtie v0.12.7 (Langmead et al., Genome Biol 2009) and quantify fractional methylation in CG, CHG, and CHH sequence contexts. Paired micrococcal nuclease sequencing reads were mapped with Bowtie 2 beta 5 (Langmead and Salzberg, Nat Methods 2012), and only nucleosomal (or similarly sized naked DNA) fragments of length 125 to 171 bp (inclusive) were analyzed. Genomic maps per base of nucleosomal fragment centers and coverage were generated with genomeCoverageBed from bedtools (http://bedtools.googlecode.com/). RNA sequencing reads were mapped with Bowtie v0.12.7 / Tophat v1.4.1 (Trapnell, Pachter, and Salzberg, Bioinformatics 2009) and FPKMs (fragments per kilobase transcript per million mapped reads) were calculated with Cufflinks v1.3.0 using bias correction (Roberts et al., Genome Biol 2011).
Supplementary files format and content: Bisulfite sequencing GFF files contain fractional methylation data for individual cytosines in either the CG, CHG, or CHH sequence context. RNA sequencing GTF files contain transcripts quantified by Cufflinks. MNase sequencing "centers" GFF files contain the number of nucleosomal fragment centers at each position and "coverage" GFF files contain the number of nucleosomal fragments covering each genomic position.
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| Submission date |
May 07, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Jason T Huff |
| Organization name |
University of California, Berkeley
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| Department |
Plant & Microbial Biology
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| Lab |
Daniel Zilberman
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| Street address |
211 Koshland Hall
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| City |
Berkeley |
| State/province |
CA |
| ZIP/Postal code |
94720 |
| Country |
USA |
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| Platform ID |
GPL17125 |
| Series (1) |
| GSE46692 |
Dnmt1-Independent CG Methylation Contributes to Nucleosome Positioning in Diverse Eukaryotes |
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| Relations |
| BioSample |
SAMN02138504 |
| SRA |
SRX275717 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1134619_Fcyl_RNAseq_transcripts.gtf.gz |
1.1 Mb |
(ftp)(http) |
GTF |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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