Data from: An indexed, mapped mutant library enables reverse genetics studies of biological processes in Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii is a leading single-celled model for dissecting biological processes in photosynthetic eukaryotes. However, its usefulness has been limited by difficulties in obtaining mutants in genes of interest. To allow generation of large numbers of mapped mutants, we developed high-throughput methods which: (1) Enable easy propagation on agar and cryogenic maintenance of tens of thousands of C. reinhardtii strains; (2) Identify mutant insertion sites and physical coordinates in such collections; (3) Validate the insertion sites in pools of mutants by obtaining >500 bp of flanking genomic sequences. We used these approaches to construct a stably maintained library of 1,935 mapped mutants, representing disruptions in 1,562 genes. We further characterized randomly selected mutants, and found that 33 out of 44 insertion sites (75%) could be confirmed by PCR, and 17 out of 23 mutants (74%) contained a single insertion. To demonstrate the power of this library for elucidating biological processes, we analyzed the lipid content of mutants disrupted in genes encoding proteins of the algal lipid droplet proteome. This study revealed a central role of the long-chain acyl-CoA synthetase LCS2 in the production of triacylglycerol from de novo synthesized fatty acids.Supplemental Data Set 1. Structural differences in the CC-4533 genome relative to reference and other strains.tpc00465_SupplementalDS1.xlsxSupplemental Data Set 2. High-confidence genomic differences unique to CC-4533.tpc00465_SupplementalDS2.xlsxSupplemental Data Set 3. Binary codes for plate super-pooling.tpc00465_SupplementalDS3.xlsbSupplemental Data Set 4. Binary codes for colony super-pooling.tpc00465_SupplementalDS4.xlsbSupplemental Data Set 5. Read counts of each flanking sequence in each super-pool.tpc00465_SupplementalDS5.xlsbSupplemental Data Set 6. Validation of flanking sequence deconvolution in randomly selected mutants.tpc00465_SupplementalDS6.xlsbSupplemental Data Set 7. Full library data: colony positions, insertion sites, deconvolution and LEAP-Seq summary, gene annotation.tpc00465_SupplementalDS7.xlsbSupplemental Data Set 8. LEAP-Seq read details for each flanking sequence.tpc00465_SupplementalDS8.txtSupplemental Data Set 9. Summary of PCR results to check the insertion sites of randomly-picked mutants from the mutant library.tpc00465_SupplementalDS9.xlsxSupplemental Data Set 10. Statistically significant insertion hotspots and coldspots.tpc00465_SupplementalDS10.xlsbSupplemental Data Set 11. Insertions in genes encoding lipid droplet proteins were characterized by PCR.tpc00465_SupplementalDS11.xls