Evaluating bias-reducing protocols for RNA sequencing library preparation
Evaluating bias-reducing protocols for RNA sequencing library preparation
The RNA fragment pool 5' - GCAGUUGCCANNNNNNNNNN  - 3' was synthesised (ThermoScientific Molecular Biology). Fragments contained both 5'phosphate and 3'hydroxyl moieties necessary for ligation. 40 ng RNA pool (~6 pmol) and 100 ng Universal Cloning Linker (~18 pmol; NEB) were denatured at 80oC for 2 min then placed immediately on ice.Ligation with 20 pmol Mth K97A was performed in 1X NEB1 buffer and 20 U SuperaseIn, at 65oC for 1 hr. Reactions were terminated by heat inactivation at 90oC and RNA/DNA recovered by isopropanol precipitation with 1 µl GlycoBlue (Ambion) as a coprecipitant. Reaction mixtures were denatured at 80oC in an equal volume of formamide loading buffer (96% Formamide, 10 mM EDTA, 0.01 % bromophenol blue, 0.01 % xylene cyanol) and separated on a 15% TBE-Urea polyacrylamide gel (Invitrogen). 250 ng small RNA marker (Abnova), denatured the same way, was run in adjacent wells. Gels were stained with SybrGold (Invitrogen) and visualised using a Safe Imager (Invitrogen) to guide excision of the 37 nt band. RNA/DNA was recovered from the gel slice by overnight elution in 300 mM NaCl, 0.1% SDS at 4oC followed by isopropanol precipitation and resuspension in 10 μl nucleasefree water. 2 µl of 1.25 µM Ion Torrent compatible reverse transcription (RT) primer (5'CGCCTTGGCC/Sp/CACTCA/Sp/CCTCTCTATGGGCAGTCGGTGATATCTATTGATGGTGCCTACAG  - 3'where Sp is an 18-atom hexa-ethyleneglycol spacer) was added to each sample, denatured at 80oC for 3 min then snap cooled on ice. Reverse transcription was performed using 200U Superscript III (Invitrogen) at 48oC for 30 min in 1X first strand buffer (Invitrogen), 0.5 mM dNTPs, 5 mM DTT and 20 U SuperaseIn. RNA was hydrolysed by incubation at 98oC for 20 min with 100 mM NaOH, 50 mM EDTA. cDNA was recovered by isopropanol precipitation and separated on a 6% TBE-Urea polyacrylamide gel. The upper band (~80bp) was excised, being careful to avoid the lower weight bands (unincorporated RT primer and RT primer dimers). cDNA was recovered from the gel slice and circularised with 100 U Circligase (Epicentre) in 1X Circligase buffer, 2.5 mM MnCl2, 0.5 mM ATP, at 60oC for 1 hr. Reactions were heat inactivated at 80oC and cDNA precipitated.
Experimental Factor: protocol type
CircLig Mth K97A