E coli dna ligase

RNA and DNA for each individual soil sample (in total, 27 soil samples) was co-extracted using the RNA PowerSoil® Total RNA Isolation Kit and the RNA PowerSoil® DNA Elution accessory kit (MoBio, CA, USA) respectively, following the manufacter’s recommendations. Extracted RNA was treated with DNase I (Roche, Germany) and RNase Out Recombinant Ribonuclease Inhibitor (Invitrogen, CA, USA) and the absence of DNA was confirmed by the lack of amplification of 16S rRNA gene, according to Villadas et al.^{54 (link)} cDNA was synthesized using random primers and SuperScript^TM II Reverse Transcriptase (Invitrogen, CA, USA) as recommended by the manufacturer. Double stranded cDNA was synthesized using RNase H (Roche), DNA polymerase I (Promega) and E. coli DNA ligase (Invitrogen, CA, USA). Blunt-end DNA was synthesized using T4 DNA polymerase (Invitrogen, CA, USA). The quantity of obtained RNA and DNA per g of soil was circa 1.0 μg and 14.8 μg, respectively as measured by Qubit 3.0 (Invitrogen, California).

Total RNA was extracted from each sample with an RNeasy Plant Mini kit (Qiagen), in which the first reagent of the kit (Buffer RLC) was added to the frozen tissue, and ground using a mortar and pestle. For each stage/tissue, 50 μg of total RNA were sent to the Joint Genome Institute (JGI). At JGI, mRNA was purified from total RNA using Absolutely mRNA™ purification kit (Stratagene) and chemically fragmented to 200-250bp (Ambion). mRNA was reverse transcribed with SuperScript II using random hexamers. Second strand cDNA was synthesized using dNTP/dUTP mix (Thermo Scientific), E. coli DNA Ligase, E. coli DNA polymerase I, and E coli RnaseH (Invitrogen). The fragments were treated with end-repair, A- tailing, and ligation of adaptors using the Illumina Truseq DNA Sample Prep Kit (Illumina). Second strand cDNA was removed by AmpErase UNG (Applied Biosystems) to generate strandedness similar to the method described by Parkhomchuk et al. [29 (link)] and enriched with 10 cycles of PCR to generate the final library. qPCR was used to determine the concentration of the libraries. Libraries were sequenced on the Illumina Hiseq, producing paired end reads R1 and R2 from each sample (fastq) with 100 bp in each read.

The eluted cDNA:mRNA hybrids (15 μl) were combined with a second-strand mix [2 μl of 5 × First-Strand Buffer (Invitrogen), 2.31 μl of Second Strand Buffer (Invitrogen), 0.23 μl of 10 mM dNTPs (NEB), 0.08 μl of 10 U/μl E. coli DNA ligase (Invitrogen), 0.3 μl of 10 U/μl E. coli DNA polymerase (Invitrogen) and 0.08 μl of 2 U/μl E. coli RNaseH (Invitrogen)] and incubated for 2 h at 16 °C. The double-stranded cDNAs were purified with AMpure XP beads (Beckman Coulter) and eluted with H₂O.