poly a polymerase tailing kit  (Lucigen Corp)

Journal: RNA Biology

Article Title: 6S-1 pRNA 9-mers are a prominent length species during outgrowth of Bacillus subtilis cells from extended stationary phase

doi: 10.1080/15476286.2025.2484519

Figure Lengend Snippet: Variation of conditions for polyC-tailing of an RNA 8- or 14-mer. (A) In lane 1, the synthetic 6S–1 pRNA 8-mer (5’-GUU CGG UC-3’; 1 µm) containing trace amounts of the sequence-identical 5’- 32 P-labelled pRNA (10,000 Cherenkov cpm) was incubated for 2 h at 37°C with 1 mM CTP, 4 mM DTT and 5 units of polyA polymerase (NEB) in 1× reaction buffer (50 mM Tris-HCl, 250 mM NaCl, 10 mM MgCl 2 , ~pH 8.0); the other lanes differed either by the CTP concentration (3 mM in lanes 3 and 4; no CTP in lanes 7 and 8) or by addition of MnCl 2 at the indicated concentrations. (B) Variation of incubation under the same conditions as in panel A, but additionally containing 2.5 mM MnCl 2 ; CTP was omitted in lane 4 and polyA polymerase in lane 5; 2 + 2 h: after 2 h at 37°C, another 5 units of polyA polymerase were added followed by incubation for another 2 h at 37°C. (C) Comparison of polyA- and polyC-tailing for the synthetic 6S–1 pRNA 14-mer (5’-GUU CGG UCA AAA CU-3’). The reaction setup was as in panel A, but using 1 mM ATP in lanes 1 and 2; 2.5 mM MnCl 2 was added in lanes 2 and 4–6. Incubation conditions were: lanes 1–4, 2 h at 37°C; lane 5, 4 h at 37°C; lane 6, 2 h at 37°C, followed by addition of another 5 units of polyA polymerase and incubation for another 2 h at 37°C. M, the 5’- 32 P-labelled 14-mer used as length marker. (D) PolyC-tailing of the 8-mer and 14-mer, but using polyA polymerase (PAP) from Cellscript or Lucigene. Reactions conditions were as in panel A, lane 2, with enzyme omission controls in lanes 3 and 4; incubation was for 2 h at 37°C. (E) Variation of MnCl 2 and CTP concentrations in polyC-tailing of the 8-mer by polyA polymerase from Lucigene. Otherwise, conditions were identical to panel D, lane 2; incubation was for 2 h at 37°C. All images show 20% PAA (acrylamide/bisacrylamide 24:1)/8 M urea gels. The gels in panels A-C were 24 cm long, 16 cm wide and 1 mm thick, those in panels D and E 20 cm long, 30 cm wide and 1 mm thick. The bottom of the gel pockets is indicated for each gel. Additional RNA size markers at the right margins of panels A, B, D and E were inferred from other 20% PAA/8 M urea gels of the same type, on which we separated RNAs of defined length together with an 8- or 14-mer. The swung dashes thus indicate that the positions of these size markers are approximations.

Article Snippet: For polyC-tailing, we used E. coli polyA polymerase (NEB), the PolyA Polymerase Tailing Kit (Lucigen) and the A-Plus PolyA Polymerase Tailing Kit (Cellscript) in the basic 1× reaction buffer containing 50 mM Tris-HCl, 250 mM NaCl, 10 mM MgCl 2 (~pH 8.0) supplemented with 4 mM DTT.

Techniques: Sequencing, Incubation, Concentration Assay, Comparison, Marker

Journal: RNA Biology

Article Title: 6S-1 pRNA 9-mers are a prominent length species during outgrowth of Bacillus subtilis cells from extended stationary phase

doi: 10.1080/15476286.2025.2484519

Figure Lengend Snippet: Read numbers of RNA-Seq libraries. Library reads were selected according to the given selection criteria as a measure of library quality. Listed are the total read numbers and the numbers of reads after selection, including the percentage of selected relative to total reads.

Article Snippet: For polyC-tailing, we used E. coli polyA polymerase (NEB), the PolyA Polymerase Tailing Kit (Lucigen) and the A-Plus PolyA Polymerase Tailing Kit (Cellscript) in the basic 1× reaction buffer containing 50 mM Tris-HCl, 250 mM NaCl, 10 mM MgCl 2 (~pH 8.0) supplemented with 4 mM DTT.

Techniques: Selection

Journal: RNA Biology

Article Title: 6S-1 pRNA 9-mers are a prominent length species during outgrowth of Bacillus subtilis cells from extended stationary phase

doi: 10.1080/15476286.2025.2484519

Figure Lengend Snippet: RNA-Seq analysis of pRNAs derived from B. subtilis 6S–1 RNA during cell outgrowth from extended stationary phase. (A–D) Length distribution (3’-end variants) of 6S–1 pRNAs in the library constructed via 3’-adapter ligation, and in polyA-, polyC 1 - and polyC 2 -tailing libraries. For details, see text and . (E, F) Numerical fit of read lengths by merging the read profiles for the polyA library with that of the polyC 1 or polyC 2 library, as described in Material and methods, illustrated in and detailed in ‘Supplementary Tables pRNA’. The y-axes depict the fraction of reads for the different pRNA length variants in the respective libraries (after read selection, see ). The read number for the most prominent bar is indicated in each profile. All 6S–1 pRNA length variants have identical 5’-ends. Error bars indicate statistical uncertainty of the fitting algorithm.

Article Snippet: For polyC-tailing, we used E. coli polyA polymerase (NEB), the PolyA Polymerase Tailing Kit (Lucigen) and the A-Plus PolyA Polymerase Tailing Kit (Cellscript) in the basic 1× reaction buffer containing 50 mM Tris-HCl, 250 mM NaCl, 10 mM MgCl 2 (~pH 8.0) supplemented with 4 mM DTT.

Techniques: RNA Sequencing, Derivative Assay, Construct, Adapter Ligation, Selection

Journal: RNA Biology

Article Title: 6S-1 pRNA 9-mers are a prominent length species during outgrowth of Bacillus subtilis cells from extended stationary phase

doi: 10.1080/15476286.2025.2484519

Figure Lengend Snippet: Length distribution for 6S–1 pRNA reads in the polyA, polyC 1 and polyC 2 libraries. Italic letters in the sequence indicate unresolved nucleotide positions in the context of the respective tailing method. The length distributions are normalized to account for library size differences for each tailing method, such that the sum of all read counts equals one. For example, for 6S–1 pRNA 8- to 12-mers in the polyA library, the normalized count is 488/581 = 0.8399 = 83.99%.

Article Snippet: For polyC-tailing, we used E. coli polyA polymerase (NEB), the PolyA Polymerase Tailing Kit (Lucigen) and the A-Plus PolyA Polymerase Tailing Kit (Cellscript) in the basic 1× reaction buffer containing 50 mM Tris-HCl, 250 mM NaCl, 10 mM MgCl 2 (~pH 8.0) supplemented with 4 mM DTT.

Techniques: Sequencing

Journal: RNA Biology

Article Title: 6S-1 pRNA 9-mers are a prominent length species during outgrowth of Bacillus subtilis cells from extended stationary phase

doi: 10.1080/15476286.2025.2484519

Figure Lengend Snippet: Schematic representation of the numerical fit workflow applied to the distribution of 6S–1 pRNA length variants. The algorithm iterates over the target sequence, from the 3’ end to the 5’ start, and differentiates between four modes, unambiguous case #1 and ambiguous cases #2–4. The four cases are defined and explained under Materials and methods, paragraph ‘Bioinformatics’. The sequence of native pRNA length variants is given from position 5 to 15; green residues indicate unambiguous positions and dark red and orange residues ambiguous positions. In the schematic diagram at the top, question marks indicate unknown position-specific read counts due to A or C trimming of reads; + sign: either unambiguous read counts (cases #1, here G5 and G6) or read counts assigned to the nucleotide 5’ of trimmed nucleotides (e.g. C8 preceding A9–A12 in the polyA library or U7 preceding C8 in the polyC 1 library) in ambiguous cases #2 to #4; SF, scaling factor. The coloured boxes embrace nucleotides linked to tailing nucleotide(s) in the polyA (magenta) or polyC1 (cyan) library, merged according to cases #1–4. The merged f 1000 values based on combined utilization of the polyA and polyC 1 libraries are shown at the bottom on the left; the merged read counts at the bottom on the right are obtained by dividing the values for ‘merged f 1000 ’ by 1000 and multiplying with the mean of the sum of read counts from both libraries, (581 + 1530)/2 = 1055.5. As an example, the merged read counts for A9 are (324.84/1000) × 1055.5 = 342.87 = ~343. For more details, see ‘Supplementary Tables pRNA’.

Article Snippet: For polyC-tailing, we used E. coli polyA polymerase (NEB), the PolyA Polymerase Tailing Kit (Lucigen) and the A-Plus PolyA Polymerase Tailing Kit (Cellscript) in the basic 1× reaction buffer containing 50 mM Tris-HCl, 250 mM NaCl, 10 mM MgCl 2 (~pH 8.0) supplemented with 4 mM DTT.

Techniques: Sequencing

Journal: RNA Biology

Article Title: 6S-1 pRNA 9-mers are a prominent length species during outgrowth of Bacillus subtilis cells from extended stationary phase

doi: 10.1080/15476286.2025.2484519

Figure Lengend Snippet: Merging of read counts for 6S–1 pRNAs from polyA and polyC 1 libraries. (A) Schematic sequence diagram for native pRNA length variants from position 5 to 15. Question marks: unknown position-specific read counts due to A or C trimming of reads; + sign: either unambiguous read counts (cases #1, here G5 and G6) or read counts assigned to the nucleotide 5’ of trimmed nucleotides (e.g. C8 preceding A9-A12 in the polyA library or U7 preceding C8 in the polyC 1 library) in ambiguous cases #2 to #4; SF, scaling factor. (B) Mutated pRNA sequence where the sequence was changed from C8 to U8 and the 200 read counts for U7/C8 in the polyC 1 library arbitrarily distributed between U7 and U8 (10 assigned to U7, 190 to U8).

Article Snippet: For polyC-tailing, we used E. coli polyA polymerase (NEB), the PolyA Polymerase Tailing Kit (Lucigen) and the A-Plus PolyA Polymerase Tailing Kit (Cellscript) in the basic 1× reaction buffer containing 50 mM Tris-HCl, 250 mM NaCl, 10 mM MgCl 2 (~pH 8.0) supplemented with 4 mM DTT.

Techniques: Sequencing