research d4004 1 l  (Zymo Research)

Journal: Methods (San Diego, Calif.)

Article Title: Multiplexed Primer Extension sequencing: a targeted RNA-seq method that enables high-precision quantitation of mRNA splicing isoforms and rare pre-mRNA splicing intermediates

doi: 10.1016/j.ymeth.2019.05.013

Figure Lengend Snippet: Material & reagents required for amplification and processing of array synthesized 1 st strand synthesis primers

Article Snippet: Materials and Reagents table ft1 table-wrap mode="anchored" t5 Table 2 caption a7 Reagent Stock Concentration Vendor Cat # Dyna Beads MyOne Streptavidin C1 10 mg/mL ThermoFisher 65001 2x Bind and Wash Buffer 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 2 M NaCl 1x Bind and Wash Buffer 5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl 1x SSC 150 mM NaCl, 15 mM Sodium Citrate Denaturing Solution 0.1 M NaOH TE Buffer 10 mM Tris pH 7.5, 1 mM EDTA Bead Elution Buffer 95% Formamide, 10 mM EDTA, pH 8.2 Open in a separate window Materials & reagents required for streptavidin bead purification table ft1 table-wrap mode="anchored" t5 Table 3 caption a7 Reagent Stock Concentration Vendor Cat # Binding Buffer 2 M Guanidinium-HCl, 75% Isopropanol Washing Buffer 10 mM TRIS pH 8.0, 80% Ethanol Open in a separate window Materials & reagents required for nucleic acid column purification table ft1 table-wrap mode="anchored" t5 Table 4 caption a7 Reagent Stock Concentration Vendor Cat # PCR Primers (oHX093 & oHX094) * 100 μM & 10 μM IDT Phusion polymerase 100x NEB M0530S Phusion buffer 5x NEB M0530S Array synthesized oligos LC Sciences DMSO 100% dNTP mix 10 mM each (dATP, dGTP, dCTP, dTTP) Sap1 restriction enzyme NEB R0569S Cutsmart buffer 10x NEB R0569S Lambda exonuclease NEB M0262S Lambda exonuclease buffer 10x NEB M0262S Isopropanol 100% Ethanol 70% Sodium Acetate 3 M, pH 5.3 Zymo DNA binding buffer Zymo Research D4003-1-L Open in a separate window * Primer sequences can be found in Table 12 Material & reagents required for amplification and processing of array synthesized 1 st strand synthesis primers table ft1 table-wrap mode="anchored" t5 Table 5 caption a7 Reagent Stock Concentration Vendor Cat # 10x Fragmentation Buffer 100 mM ZnCl 2 , 100 mM Tris-HCl pH 7.0 Fragmentation stop buffer 0.5 M EGTA Open in a separate window Materials & reagents required for RNA fragmentation table ft1 table-wrap mode="anchored" t5 Table 6 caption a7 Reagent Stock Concentration Vendor Cat # SSIV RT Buffer 5x ThermoFisher 18090050 Superscript IV ThermoFisher 18090050 1 st strand primer pool Each primer in S. cerevisiae pool at ~80 nM (309 primers).

Techniques: Amplification, Synthesized, Concentration Assay, Binding Assay