For the EhAGO2 2 IP RNA library, RNA was extracted from the IP material, directly ligated to the 30 adapter oligonucleotide, size fractionated find more information and treated with CIP and PNK, and then ligated to the 5�� adapter oligonucleotide. Inhibitors,Modulators,Libraries For the Rah man small RNA library, 100 ug of small RNA enriched RNA was size fractionated on a 12% TBE urea polyacryl amide gel, the 15 30nt fraction was excised, followed by 30 adapter ligation, CIP PNK treatment, and 50 adapter ligation as above. For both libraries, the final ligated RNA with both 50 and 3�� adapters was converted to single stranded cDNA using Superscript II reverse transcriptase. The cDNA was PCR amplified using 454 Pri mers for 20 cycles, and resolved on a 4% low melting point agarose gel. The band at 100 bp was ex cised and purified, and then further heat denatured and purified from a 6% PAGE urea gel.
After a second round of purification, the recovered DNA was resuspended in Qiagen elution buffer, and pipelined into the 454 sequen cing procedure. Small RNA read processing Inhibitors,Modulators,Libraries and mapping All sequencing reads were processed by first removing the linker from both ends. the resulting sequences were analyzed with Unix tools and unique sequences selected. The unique reads Inhibitors,Modulators,Libraries were mapped against the E. histolytica HM 1 IMSS genome, release 1. 3, using the program Bowtie with parameters set as v 1, k 5. Mapped reads were visualized with the genome browser IGV. To identify small RNAs that map to exon exon junctions, TopHat was used with the following parameters report sec ondary alignments G Ehistolytica AmoebaDB 1. 3. gtf i 20.
For the scaffold view of mapped small RNAs, histo Inhibitors,Modulators,Libraries grams were generated in R using a window size of 500 bp to divide the scaffolds. For tRNA and rRNA analysis, we downloaded all tRNA array sequences from NCBI based on previously published ana lysis. For repetitive element analysis, we made a cus tom dataset using all SINEs/LINEs/EhEREs coordinates recently deposited to AmoebaDB, then aligned all sequences with Bowtie using the parameters v 2, k 5. All sequences from both small RNA libraries have been deposited at AmoebaDB The small RNA sequence data from this study have been submitted to the NCBI Gene Expression Omnibus under accession num ber GSE43668. Nucleotide composition of small RNAs and distribution of small RNAs on gene loci For each subset of small RNA populations the small RNA read sequences were extracted.
The nucleotide com position at each position was counted using the R package ShortRead, and frequency bar plots generated from these data. For small RNA distri bution on gene loci, Inhibitors,Modulators,Libraries only genes with 50 small RNAs mapping were considered. kinase inhibitor Y-27632 Each small RNA sequence was assigned a position value based on the position of the starting nucleotide along the gene. Histo grams for these values were plotted in R. Small RNA Northern blot analysis Small RNA Northern blot analysis was done as previ ously published.