EF-1 alpha ribonomics- Stress 2
To identify mRNAs associated with EF-1a protein or complexes, rabbit anti-EF-1a antibody (15 µl) was incubated with 50 µl of goat anti-rabbit magnetic beads (New England Biolabs) and 40 U/ml RNAse OUT (Invitrogen) for 16 hours at 4°C with shaking. Cytoplasmic extract corresponding to 1 x 109 cells was then incubated with the beads. The beads were then washed three times with IMP1 buffer. The immunoprecipitated RNAs were eluted and purified with the RNeasy® (Qiagen) kit by using the “Animal Cells I” protocol in the manufacturer’s manual but with an additional column-based DNase treatment step. The purified RNAs were then subjected to deep sequencing using the SOLiD 3 pus platform.   The sequencing data obtained were analyzed using the CLC Genomics Workbench© v 5.5.1. The reads were trimmed on the basis of quality using a threshold phred score of 15. The T. cruzi reference genome used for mapping was obtained from the NCBI database (AAHK01), and the alignment was performed with the following parameters: additional upstream and downstream sequences of 100 bases; minimum number of reads, 10; maximum number of mismatches, 2; nonspecific match limit, -2; use of colorspace encoding. The minimum similarity of the reads mapped to the reference genome was 70%. We selected possible targets of EF-1a mRNPs by using the beta binomial statistical test [43], with a p-value-corrected FDR of = 1% and a minimum four-fold change with respect to the control (pre-immune serum) as the threshold for significance.