GSM1134623: O. lucimarinus bisulfite sequencing; Ostreococcus 'lucimarinus'; Bisulfite-Seq
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) was 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 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). 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). For MNase sequencing, 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). Bisulfite-Seq, RNA-Seq and MNase-Seq