Scriptseq kit

Duplicate cultures of the M3 GAS strains MGAS10870, 10870ΔrocA, and 10870::rocA^M1 were grown to the exponential phase of growth and RNA was isolated as described above. Ribosomal RNAs (rRNAs), which represent ~96% of total RNA, were depleted using the Ribo-Zero Gram-Positive rRNA Removal Kit (Epicentre). The rRNA-depleted RNA was used to generate libraries for sequencing using the ScriptSeq kit (Epicentre), as we have previously described (McClure et al., 2013 (link)). Briefly, RNA was fragmented, cDNA was synthesized using random hexamers containing a 5′ tagging sequence, the RNA was hydrolyzed, the cDNA was tagged at the 3′ end, a limited number of PCR cycles (n=14) were used to amplify the libraries via the 5′ and 3′ tags (the libraries will be barcoded by using different primers), and the libraries were size-selected (170 to 300 bp). The six size-selected and barcoded libraries were ran on an Illumina MiSeq flowcell. Data were analyzed using CLC Genomics Workbench and normalized to overall sequencing depth using total mapped reads data. Statistical significance was tested using Kal’s Z-test with a false-discovery rate correction (Kal et al., 1999 (link)). The RNAseq data have been deposited at the Gene Expression Omnibus (GEO) database at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/geo) and are accessible through accession number GSE68277.

hrr25is was grown to OD₆₀₀ ~0.3 at 30°C in YPD. Cells were treated with either DMSO or 20μM CMK (MedChem Express) for 1hr, were harvested, and total RNA was extracted with hot phenol^{52 (link)}. 50 μg total RNA was treated with DNase (Invitrogen) and rRNA was depleted using Ribo-Zero beads (Epicentre). RNA was fragmented, reverse transcribed, and barcoded using the ScriptSeq kit (Epicentre). RNA-seq libraries were sequenced on an Illumina HiSeq 2500 System. 100 bp single-end reads were obtained, adapter sequences were clipped, and reads were mapped to the S288c genome using STAR^{53 (link)}. HTSeq was used to count reads^{54 (link)}, and DESeq^{55 (link)} was used to analyze differential expression among two replicates. Traces from RNA-seq were from a single replicate. Read coverage was normalized in deepTools^{56 (link)} using –normalizeUsingRPKM and plotted. 3ʹ extension indices were calculated using the RPKM-normalized reads using an average of both replicates.