title: GSM1684589: Chlamydomonas reinhardtii, replicate 3 (4A+, log phase); Chlamydomonas reinhardtii; Bisulfite-Seq
library_construction_protocol: C. reinhardtii wild type strain 4a+ were cultured using Tris-acetate-phosphate (TAP) medium in Erlenmeyer flasks at 25 degrees Celsius with agitation at 180 rpm under light irradiation at 100 microE. Cells were harvested at mid-log phase (at approximately 2 x 106 cells/ml). Cells were lysed using lysis buffer (50mM Tris-HCl pH 8, 200mM NaCl, 2mM EDTA, 2% SDS and proteinase K) and genomic DNA was extracted using CTAB-based protocol39. 5ug of gDNA was sheared to 700bp using the focused-ultrasonicator S2 (Covaris) and spiked in with 50ng HaeIII digested lambda DNA for quality control. The fragments were treated with end-repair, A- tailing, and ligation of methylated Illumina adaptors (Illumina) using the DNA library preparation kit for Illumina (Kapa Biosystems). The adaptor ligated DNA was subjected to bisulfite treatment using EpiTect Bisulfite Kit (Qiagen). The bisulfite-treated DNA was amplified with 8 cycles of PCR with uracil-insensitive Taq polymerase (Pfu Turbo Cx Hotstart DNA polymerase; Stratagene). The PCR product was run on a 2% agarose gel followed by gel excision to select for fragments between 300 to 400bp. Purified library was sequenced on the Illumina Hiseq.
attributes.tag: GEO Accession
attributes.value: GSM1684589