Journal: Genome Biology

Article Title: Efficient and robust RNA-seq process for cultured bacteria and complex community transcriptomes

doi: 10.1186/gb-2012-13-3-r23

Figure Lengend Snippet: Performance evaluation of five rRNA depletion methods. (a) Shown is the distribution of RNA-seq reads aligning to protein-coding sequences (CDS; blue), rRNA (red), and other regions (tRNA, non-coding RNA, small RNA, and intergenic regions; gray) for undepleted total RNA (top) and five rRNA depletion protocols. (b) The lengths of the black bars represent the coefficient of determination (R2) for RPKM values before and after rRNA depletion using different rRNA-depletion methods. Ribo-Zero, normalization using duplex-specific nuclease (DSN) and Ovation were tested on a 1:1:1 pool (by mass) of total RNA prepared from P. marinus, E. coli, and R. sphaeroides. MICROBExpress and mRNA-ONLY were performed on individual RNA preparations without pooling.

Article Snippet: The second strand was synthesized by adding 1× of second strand buffer (5×; Invitrogen), 0.2 mM of dNTPs (10 mM; Invitrogen), 40 U of E. coli DNA polymerase I (10 U/μl; NEB, Ipswich, MA, USA), 10 U of E. coli DNA ligase (10 U/μl; NEB), 5 U of RNase H (5 U/μl; Invitrogen) to the first strand reaction (150 μl total volume).

Techniques: RNA Sequencing Assay

Journal: Genome Biology

Article Title: Efficient and robust RNA-seq process for cultured bacteria and complex community transcriptomes

doi: 10.1186/gb-2012-13-3-r23

Figure Lengend Snippet: Depletion of rRNA in a mixture of total RNAs from E. coli, R. sphaeroides and P. marinus with Ribo-Zero is reproducible and works well with fragmented total RNA. (a) The pie charts represent the mapped read distributions of protein-coding genes (CDS; blue), rRNA (red), and other reads (tRNA, non-coding RNA, small RNA and intergenic regions; gray) for undepleted total RNA, two technical replicates of Ribo-Zero treatment of intact total RNA and for Ribo-Zero treatment of fragmented total RNA. (b,c) Double-log scatter plots of RPKM values and the coefficient of determination (R2) for the technical Ribo-Zero replicates (b) and for Ribo-Zero treatment of fragmented versus intact total RNA (c). Points on the axes represent CDSs with zero coverage in one of the two samples. The number of data points in the diagonal cloud and on the axes is indicated. The total number of annotated CDSs in the three bacterial genomes is 10,278.

Article Snippet: The second strand was synthesized by adding 1× of second strand buffer (5×; Invitrogen), 0.2 mM of dNTPs (10 mM; Invitrogen), 40 U of E. coli DNA polymerase I (10 U/μl; NEB, Ipswich, MA, USA), 10 U of E. coli DNA ligase (10 U/μl; NEB), 5 U of RNase H (5 U/μl; Invitrogen) to the first strand reaction (150 μl total volume).

Techniques:

Journal: Genome Biology

Article Title: Efficient and robust RNA-seq process for cultured bacteria and complex community transcriptomes

doi: 10.1186/gb-2012-13-3-r23

Figure Lengend Snippet: Strand specificity of RNA-seq reads. Shown is a 17-kb window of the E. coli genome viewed with the Artemis browser [28]. The mapped reads aligning to the top strand (green) or bottom stand (purple) consistent with the direction of the annotated genes as represented by the blue boxes with arrows and corresponding gene ID numbers and operons below (for example, genes b3196 through b3206).

Article Snippet: The second strand was synthesized by adding 1× of second strand buffer (5×; Invitrogen), 0.2 mM of dNTPs (10 mM; Invitrogen), 40 U of E. coli DNA polymerase I (10 U/μl; NEB, Ipswich, MA, USA), 10 U of E. coli DNA ligase (10 U/μl; NEB), 5 U of RNase H (5 U/μl; Invitrogen) to the first strand reaction (150 μl total volume).

Techniques: RNA Sequencing Assay