Bulked segregant analysis of Chlamydomonas reinhardtii: multicellular pool
Pools of WRC01 x CC-125 F1 progeny were crossed to generate a large pool of F2 progeny for use in bulked segregant analysis. We separated F2 progeny into primarily unicellular and primarily multicellular pools by four rounds of growth and centrifugation. 10 mL of pooled algae were transferred to a 15 mL conical tube and centrifuged for 1 min. at 100 x g. The top 2 mL (for the unicellular pool) or the bottom 1 mL (for the multicellular pool) was used to inoculate 50 mL TAP medium. We extracted total DNA from unicellular and multicellular pools using the DNeasy Plant Mini Kit (Qiagen). DNA libraries were generated by the Vincent J. Coates Genomics Sequencing Laboratory (Berkeley, CA) using the PrepX ILM 32i DNA Library Kit and were sequenced on an Illumina HiSeq 2500 at the Georgia Institute of Technology.