Journal: Nature protocols

Article Title: Mapping the miRNA interactome by c ross l inking l igation a nd s equencing of h ybrids (CLASH)

doi: 10.1038/nprot.2014.043

Figure Lengend Snippet: Inter-molecular RNA-RNA ligation TIMING overnight

Article Snippet: Hybond-C Extra membrane (GE Healthcare, RPN303E) Kodak BioMax MS Autoradiography Film (#8222648) MetaPhor agarose (Lonza, #50180) SYBRSafe (Life Technologies, S33102) cOmplete Protease inhibitors, EDTA-free (Roche Applied Science, #11873580001) RNace-IT (Agilent, #400720) diluted 1:20 with water, store at 4°C for at least 2 years SeeBlue Plus2 Pre-Stained Standard (Life Technologies, LC5925) NuPAGE LDS Sample Buffer 4X (Life Technologies, N0007) NuPAGE 4-12% polyacrylamide Bis-Tris gels (Life Technologies, NP0335) NuPAGE SDS MOPS running buffer (Life Technologies, NP0001) NuPage Transfer Buffer (Life Technologies, NP00061) GlycoBlue (Life Technologies, AM9515) MinElute PCR purification kit (QIAGEN, #28004) MinElute Gel extraction kit (QIAGEN, #28604) GeneRuler 50 bp DNA ladder (Thermo Scientific, SM0371) Qubit dsDNA HS Assay Kit (Life Technologies, {"type":"entrez-protein","attrs":{"text":"Q32854","term_id":"75280861","term_text":"Q32854"}} Q32854 ) 6 x DNA Loading dye (Thermo Scientific, R0611) T4 PNK, T4 Polynucleotide Kinase (New England BioLabs, M0201L) T4 RNA ligase 1 (New England BioLabs, M0204L) T4 RNA ligase reaction buffer, 10× (supplied with T4 RNA ligase 1) T4 RNA ligase 2 truncated, K227Q (New England BioLabs, M0351L) TSAP, Thermosensitive Alkaline Phosphatase (Promega, M9910) Proteinase K (Roche Applied Science, #03115836001) SuperScript III Reverse Transcriptase (Life Technologies, #18080-044) 5 x First strand buffer (supplied with SuperScript III Reverse Transcriptase) 0.1M DTT (supplied with SuperScript III Reverse Transcriptase) RNase H (New England BioLabs, M0297L) TaKaRa LA Taq (Clontech, RR002M) 10X LA PCR Buffer ll (Mg 2+ plus) supplied with TaKaRa LA Taq dNTPs 2.5mM (supplied with TaKaRa LA Taq).

Techniques: Ligation, Concentration Assay

Journal: Nature protocols

Article Title: Mapping the miRNA interactome by c ross l inking l igation a nd s equencing of h ybrids (CLASH)

doi: 10.1038/nprot.2014.043

Figure Lengend Snippet:

Article Snippet: Hybond-C Extra membrane (GE Healthcare, RPN303E) Kodak BioMax MS Autoradiography Film (#8222648) MetaPhor agarose (Lonza, #50180) SYBRSafe (Life Technologies, S33102) cOmplete Protease inhibitors, EDTA-free (Roche Applied Science, #11873580001) RNace-IT (Agilent, #400720) diluted 1:20 with water, store at 4°C for at least 2 years SeeBlue Plus2 Pre-Stained Standard (Life Technologies, LC5925) NuPAGE LDS Sample Buffer 4X (Life Technologies, N0007) NuPAGE 4-12% polyacrylamide Bis-Tris gels (Life Technologies, NP0335) NuPAGE SDS MOPS running buffer (Life Technologies, NP0001) NuPage Transfer Buffer (Life Technologies, NP00061) GlycoBlue (Life Technologies, AM9515) MinElute PCR purification kit (QIAGEN, #28004) MinElute Gel extraction kit (QIAGEN, #28604) GeneRuler 50 bp DNA ladder (Thermo Scientific, SM0371) Qubit dsDNA HS Assay Kit (Life Technologies, {"type":"entrez-protein","attrs":{"text":"Q32854","term_id":"75280861","term_text":"Q32854"}} Q32854 ) 6 x DNA Loading dye (Thermo Scientific, R0611) T4 PNK, T4 Polynucleotide Kinase (New England BioLabs, M0201L) T4 RNA ligase 1 (New England BioLabs, M0204L) T4 RNA ligase reaction buffer, 10× (supplied with T4 RNA ligase 1) T4 RNA ligase 2 truncated, K227Q (New England BioLabs, M0351L) TSAP, Thermosensitive Alkaline Phosphatase (Promega, M9910) Proteinase K (Roche Applied Science, #03115836001) SuperScript III Reverse Transcriptase (Life Technologies, #18080-044) 5 x First strand buffer (supplied with SuperScript III Reverse Transcriptase) 0.1M DTT (supplied with SuperScript III Reverse Transcriptase) RNase H (New England BioLabs, M0297L) TaKaRa LA Taq (Clontech, RR002M) 10X LA PCR Buffer ll (Mg 2+ plus) supplied with TaKaRa LA Taq dNTPs 2.5mM (supplied with TaKaRa LA Taq).

Techniques: Concentration 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: 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