RNA was DNAse treated and digested before cDNA synthesis with Eco 130I, which cuts the DNA in the middle of the amplification area in order to get rid of possible genomic contamination.
Reverse transcription is then performed using oligo dT) as a 3' adaptor and the cDNAs formed by second strand synthesis are digested with MmeI to generate 20 bp signatures that are sequenced and used to identify miRNA target pairs [ 15– 17].
Prior to cDNA synthesis RNA was digested with RNase-free DNase I (Roche Diagnostics, North America).
First strand synthesis of DNAse digested RNA and subsequent qRT-PCR experiments were performed as described earlier [ 19].
For mRNA synthesis, constructs were digested with SmaI or HindIII, and mRNAs produced using the mMessage mMachine RNA kit (Ambion, Austin, TX, USA).
DNA libraries were fractionated on a nondenaturing 1x TBE, 6% acrylamide gel (Invitrogen), and fragments between 250 and 350 bp were excised from the gel and eluted by incubation in TE at room temperature with vigorous shaking for 30 min. Uridine-containing, second-strand synthesis products were digested with Uracil-N-Glycosylase (Applied Biosystems).
The RNA was digested with shrimp DNAse before cDNA synthesis, which was performed using a mix of oligo-dT and random hexamer primers and SuperscriptII reverse transcriptase.
1 μg RNA was used as template for cDNA synthesis after digest of genomic DNA with RNase-free DNase (RevertAid First Strand cDNA synthesis Kit, Fermentas Life Science, St. Leon-Rot, Germany).
Efficiency of rumen microbial N synthesis based on OM truly digested in the rumen was enriched by U Cas supplementation.
Residues of DNA were digested using DNase I, Rnase-free (Thermo Scientific), for cDNA synthesis RevertAid First Strand cDNA Synthesis Kit was used.
Double strand cDNA was synthesized using the "Double strand cDNA synthesis kit" (Invitrogen, Carlsbad, CA) and digested with the NlaIII isoschizomer Hin1II (MBI Fermentas).
