In vivo CRAC and Blotting Analysis

In vivo CRAC, western and northern blot analyses were performed essentially as described previously (Granneman et al, 2009 (link)) with the following modifications: For the Rat1 CRAC experiments shown in Figure 6, cells were pre-grown in filter sterilized synthetic minimal medium containing galactose and raffinose, but lacking uracil and tryptophan (SG/R-URA-TRP), to an OD₆₀₀ of 1.0, subsequently shifted to glucose containing medium (SD-URA-TRP) for 12 h to an OD₆₀₀ of ∼0.5. Cultures were UV irradiated in the Megatron (Supplementary Figure S1A) at room temperature for 100 s (equivalent to an average dose of ∼1.6 mJ/cm²) and cells were harvested by centrifugation, washed with phosphate-buffered saline (PBS), and stored at −80 °C. Megatron parts were purchased from UVO3 (http://www.uvo3.co.uk; contact Peter Wadsworth http://Peter@uvo3.co.uk). Sanger sequencing of cDNAs was performed as described (Granneman et al, 2009 (link)). Negative control cDNAs were generated from RNA extracted from excised membrane fragments from mock experiments run in parallel. For the high-throughput sequencing analysis, crosslinked RNAs were sequentially ligated to L3 and barcoded L5 adaptors and amplified by RT/PCR (see Supplementary Table S1 for oligonucleotide sequences). Oligonucleotides were purchased from Integrated DNA technologies (IDT). Illumina sequencing (single end 50-bp reads) was performed according to the manufacturer's procedure and reads were aligned to the yeast genome using novoalign (http://www.novocraft.com). Data analyses were performed using pyCRAC, a set of Python tools for high throughput sequence analysis (Webb, Tollervey and Granneman, manuscript in preparation).