The cDNA-containing vectors were linearized with Xho I and used as templates for RNA synthesis using the mCAP RNA Capping Kit (Stratagene Cloning Systems, La Jolla, CA).
For cDNA synthesis, 1 μg of total RNA was reverse transcribed according to the manufacturer's instructions using the high Cap-Kit from AppliedBiosystems (ABI), Darmstadt, Germany.
Briefly, cDNA inserts from the arrayed library were amplified by PCR using the flanking primers from the Cap Finder kit and selected for spotting after a short run on a 2.5% agarose gel.
A microwave antigen-retrieval technique was used and the samples were treated in target retrieval solution, pH 9 (Dako), for 20 min at 750 W. The Cap-plus kit (Zymed, San Francisco, CA) was used for immunostaining and Mayer's hematoxylin (Sigma, St Louis, MO and Labvision, Fremont, CA) was used for counterstaining.
These two ZFN plasmids were later used as templates for the preparation of PAM1 and PAM2 mRNA, by in-vitro transcription using MessageMax T7 ARCA-Capped Transcription Kit (Cellscript).
The bovine embryonic array used here originates from a bovine cDNA library established at the ovoid stage, starting from 1.6 μg of RNA and using the Cap Finder cDNA kit from Clontech as described in Degrelle et al. [ 26].
Capped RNA was synthesised using the mMessage mMachine® Capped RNA Transcription kit (Ambion) according to the manufacturer's instructions.
Capped cyclin D1 mRNA was generated by using a mMESSAGE mMACHINE high yield capped RNA transcription kit containing T7 RNA polymerase (Ambion).
Capped luciferase mRNA with and without poly(A) tail was generated by mMESSAGE mMACHINE high yield capped RNA transcription kit containing T3 RNA polymerase (Ambion).
After purification of the linearized DNA, Cas9 and gRNA mRNA were transcribed in vitro using a MessageMAXT7 ARCA-Capped Message Transcription Kit (CELLSCRIPT, Madison, WI, USA) and a MEGAshortscript T7 Kit (Life Technologies), respectively.
