title: JP03
organism: Chlamydomonas reinhardtii
description: Protocols: Summary: This experiment used a wild-type and psr1 knockdown strain, grown at low and high phosphate, assayed at day 3 and day 5. Strains: C. reinhardtii wild type strain 137c (CCAP 11/32C or was obtained from the UK Culture Collection of Algae and Protozoa) and the psr1 knockdown strain (Shimogawara K1, Wykoff DD, Usuda H, Grossman AR. 1999. Chlamydomonas reinhardtii mutants abnormal in their responses to phosphorus deprivation. Plant Physiol. 120(3):685-94) from Jeff Moseley and Arthur Grossman (Carnegie Institution, Stanford, USA). Growth conditions: The strains were grown photoheterotrophically on a standard Tris-acetate-phosphate (TAP) medium (high phosphate media (HP)) (E.H. Harris, The Chlamydomonas Sourcebook: A Comprehensive Guide to Biology and Laboratory Use, Academic Press, San Diego (1989) p. 780) in 50 ml nunc flasks at 25 degree celsius under cool-white fluorescent light (150 micromol m-2 s-1.) with a 16-h light:8-h dark light regime. Before starting Phosphate deficient (low phosphate (LP)) growth, cells were grown photoheterotrophically in HP media to reach logarithmic phase then harvested and washed twice with LP media by centrifugation at 3000g for 1 min. Washed cells were then re-suspended in the same medium to get an initial cell count of ~65 x 103 cells ml-1. The LP media was prepared by reducing P content of HP media from 1 mM P to 0.1 mM P, potassium phosphate (pH 7.0) solution (K2HPO4/KH2PO4) was used as Phosphate source in media. The concentration of K was kept uniform in LP media by adding KCl. The pH of both HP and LP media was buffered to 7.0 using Tris. A total of 200 ml of WT and psr1 cultures was used for RNA extraction. The cell count of HP day 3 sample were approx. 2x 106 cells ml-1 and LP day 3 were approx 3.8 x 105 cells ml-1 . Day 5 cell count, HP: 4x106 cells ml-1 and LP: 11x 105 cells ml-1. growth was on high (1mM) or low (0.1mM) phosphate Total RNA was extracted using TRizole extraction method. SOLiDTM Total RNA-Seq Kit (PN 4445374)