Bovine tissue samples including heart, lung, muscle, brain, intestine and spleen were collected at a local abattoir. Bovine GV oocytes and early stage embryos were purchased from Bomed, Inc. (Madison, WI). All samples were frozen in liquid nitrogen and stored at -80 ◦C until use.
The open reading frame (ORF) of bovine KPNA7 cDNA was PCR amplified from a KPNA7 expression plasmid  and cloned into pcDNA3.1/myc-His vector (Invitrogen, Carlsbad, CA) using a forward primer containing a Kozak sequence and BamHI site and a reverse primer containing a XhoI site (Additional file 1, Figure 1). The plasmid designed to express the bovine miRNA-1296 was prepared by PCR amplification of a 291 bp genomic fragment containing the pre-miRNA-1296 followed by cloning into pcDNA3.1 vector using a forward primer containing a BamHI site and a reverse primer containing a PmeI site (Additional file 1, Figure 1). Both constructs were sequenced to ensure that no mutations were introduced during PCR amplification.
DNA samples isolated from oocytes/embryos or somatic tissues were treated by bisulfite following the manufacturer’s instructions of the EZ DNA Methylation-Direct™ kit (Zymo Research, Irvine, CA). Primers (Additional file 1, Figure 1) were designed using Methprimer online program and were used in a 25-µl PCR reaction for the first round of 40 cycles. Cycling conditions were as follows: 95°C for 9 min followed by 40 cycles of 95 °C for 30 s, 54 °C for 30 s, 72 °C for 30 s and a final extension of 5 min at 72 °C. Nested PCR of 35 cycles was performed using the PCR product as a template in a higher annealing temperature at 58 °C and a shorter denaturation time of 5 min at 95 °C. The DNA regions of CpG sites were amplified, and three independent PCR reactions were performed. The PCR products were purified, pooled together and cloned into pGEM-T Easy vector (Promega). After verification, 10 clones for each DNA sample were sequenced, and the sequences were analyzed using the online tool QUMA (http://quma.cdb.riken.jp/).
HEK293 cells were cultured in DMEM (Invitrogen, Carlsbad, CA) containing 10 % FBS. For transient transfection, Xtremegene 9 (Roche Applied Science, Indianapolis, IN) was used according to manufacturer’s instructions. Following transfection, cells were incubated for 24 h before harvest for western blot analysis. CCL22 cells were cultured in DMEM containing 10 % HS. 5-aza-2-deoxycytidine (5-Aza) was added to the culture medium with a final concentration of 0.5 µM in 6-well plates (Corning Inc., Corning, NY) seeded with cells 24 h before the treatment. Treatment continued for 3 days with fresh 5-Aza changed every 24 h. After 3 days, cells were harvested and stored at -80 °C until use.
Western blot analysis
Electrophoresis and transfer were performed according to a previous study with minor modifications . HEK293 cell were harvested with Pierce IP Lysis Buffer (Thermo Fisher Scientific, Waltham, MA), and 10 μl of cell lysate were mixed with an equal volume of Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA). Protein samples (15 μg/each) were separated on a 4-20% gradient ready gel (Bio-Rad) and transferred onto a Immobilon-FL PVDF membrane (Millipore, Billerica, MA). Following transfer and blocking in 5 % nonfat dry milk in PBS containing 0.1% Tween-20 (PBST) for one hour, the membrane was incubated in anti-Myc antibody (Sigma-Aldrich, St. Louis, MO) diluted 1:1000 and anti-ACTB antibody (GenScript, Piscataway, NJ) diluted 1:1000 in blocking buffer overnight at 4 °C. After 3 washes, two secondary antibodies IRDye 800CW goat anti-rabbit and IRDye 680LT goat anti-mouse (Li-COR, Lincoln, NE) were added to the blocking buffer and incubated for 20 min. Detection of protein was performed following the instructions of the Odyssey system (Li-COR, Lincoln, NE). ImageJ software was used to quantify the protein bands. Abundance of KPNA7 protein was normalized relative to the abundance of ACTB protein.
Quantitative real-time PCR (RT-qPCR)
cDNA from oocyte and embryo samples (n = 3 pools of five each) was prepared by lysing the samples in 1× miScript RT buffer containing 0.5 % NP-40 at 95 °C for 5 min followed by addition of miScript reverse transcriptase mix (Qiagen, Valencia, CA) and incubation at 37 °C for 60 min. The cDNA was then used for determination of relative amount of miR-1296 by RT-qPCR using the miRNA-1296 specific primer and the miScript universal primer (Qiagen, Valencia, CA). Bovine miRNA-125b was used as an endogenous control as this miRNA is expressed consistently in preimplantation embryos . RT-qPCR analysis was performed on the Bio-Rad CFX96 system. The iQ™ SYBR Green Supermix (Bio-Rad, Hercules, CA) was used in 20 μl reaction volumes containing 100 nM of each primer and 5 μl of diluted cDNA. Cycling parameters were 95 °C for 15 min, and then 40 cycles of 95 °C for 15 s, 55 °C for 30 s and 70 °C for 30 s. Standard curves for the target and control miRNA were constructed using 10-fold serial dilution of a pooled cDNA sample.