Small RNA library generation and data analysis pipeline. Total RNA was processed to enrich the sample for small-sized RNAs by two consecutive PAA gel fractionation and elution steps. The 5S rRNA was depleted by hybridization with a complementary biotinylated oligonucleotide. The small RNA fraction was C-tailed with E. coli poly(A) polymerase and reverse-transcribed using a poly(G) oligonucleotide; the cDNA obtained was subjected to 454 sequencing on the Genome Sequencer FLX System from the Lifesequencing S.L. company. The total number of reads obtained, the reads that successfully mapped to the S. pneumoniae TIGR4 genome, and the total number of cDNA contigs finally obtained (≥50 nt) are indicated. Six percent PAA gels stained with ethidium bromide show RNA from the fractionation procedure. (St) The RNA marker used.
