The sequences and locations of shRNAs are listed in Table 2. Plasmid DNA (pCMV-Script, Stratagene) encoding the HAVCR1 protein from ACHN cells was transfected into HEK-293 cells using Lipofectamine LTX (Invitrogen).
Two microgram of total RNA was reverse-transcribed to DNA using the Script cDNA Synthesis Kit (170-8891, Bio-Rad Laboratories).
I fully support the work to decode the genetic script of DNA (Science Times, March 10).
In vitro RNA was transcribed from linearized plasmid DNA using the Mega Script RNA kit from Ambion with either SP6 or T7 RNA polymerase.
To validate the microarray data, 1500 ng of DNase-treated RNA was reverse transcribed to make 50 ng/ µL of complementary DNA (cDNA) using Affinity Script qPCR cDNA synthesis kit (Agilent – Lot# 6077352).
For all the analyses, the computations themselves are straightforward and used C++ programs to interrogate the DNA sequences and R scripts for the statistical analyses.
The target DNA was a pPCR-Script Amp SK plasmid (Stratagene, Amsterdam, The Netherlands) with an insert at the Srf I site in the multiple cloning site (supplied by N.I.S.T, Gaithersburg, MD, USA).
The activity of 1 μl of Top1 was assayed in 30 μl of reaction volume containing 0.5 μg of negatively supercoiled pBlue-Script KSII DNA, that is present in both dimeric and monomeric forms and reaction buffer (20 mM Tris/HCl pH 7.5, 0.1 mM Na2EDTA, 10 mM MgCl2, 50 μg/ml acetylated BSA and 150 mM KCl).
Before submitting to Illumina for sequencing, the quality of the library was determined by cloning a portion of DNA fragments into the pPCR-Script Amp SK Cloning Vector provided in the PCR-Script Amp Cloning Kit (StrataGene, #211188), per manufacturer's instruction, with the molar ratio of insert to vector DNA ranges at 50 1.
