Escherichia coli dna pol 1

RNA sequencing libraries were constructed in parallel from the six species using TruSeq RNA Sample Prep Kits (Illumina, SanDiego, USA). Briefly, first strand cDNA synthesis was performed with oligo-dT primer and Superscript II reverse transcriptase (Invitrogen, CA, USA). The second strand was synthesized with Escherichia coli DNA Pol I (Invitrogen, CA, USA). Double-stranded cDNA was purified with a Qiaquick PCR purification kit (Qiagen), and sheared with a nebulizer (Invitrogen, CA, USA) into 200–250 bp fragments. After the end repair and addition of a 3′-dA overhang, the cDNA was ligated to Illumina PE adapter oligo mix (Illumina). The products were then purified and enriched with PCR to create the final sequencing cDNA library. Both ends of the library were sequenced on the Illumina HiSeq 2000 platform.

RNA sequencing was performed as described [26 (link)]. Briefly, poly (A) RNA was prepared from total RNA by two rounds of oligo (dT)₂₅ Dynabeads (Invitrogen) purification. Following fragmentation at 94 °C for 3.5 min, the RNA was converted to first strand cDNA using random hexamer primer and Superscript II (Invitrogen), followed by second-strand cDNA synthesis with Escherichia coli DNA pol I (Invitrogen) and RNAse H (Invitrogen). The barcoded sequencing library was prepared and sequenced on Illumina HiSeq for 1 × 100 cycles following the standard protocol.