Illumina Genome Analyzer IIx paired end sequencing; Characterization of long non-coding RNAs expressed during oocyte-to-embryo transition in mice
Characterization of long non-coding RNAs expressed during oocyte-to-embryo transition in mice
Metaphase II-arrested eggs (MII eggs) were obtained from superovulated 3-week-old C57BL6/J and B6D2F1 female mice (SLC Japan, Shizuoka) that were first injected intraperitoneally with 5 IU of equine chorionic gonadotropin (eCG; ASKA Pharmaceutical Co., Tokyo) followed 48 h later with 5 IU of human chorionic gonadotropin (hCG; ASKA Pharmaceutical). MII eggs were collected from the ampullae of oviducts 15 h post-hCG injection and transferred to human tubal fluid medium (Lawitts & Biggers, 1993) supplemented with 10 mg/ml BSA (Sigma-Aldrich, Saint Louis, MO). The eggs derived from C57BL6/J and B6D2F1 mice were inseminated with spermatozoa obtained from the caudal epididymis of adult C57BL6/J and ICR mice (SLC Japan), respectively. The spermatozoa were incubated for 2 h in TYH medium (Toyoda Y, 1971) and human tubal fluid medium supplemented with 10 mg/ml BSA for the eggs from C57BL6/J and B6D2F1 female mice, respectively, in an atmosphere of 5% CO2/95% air at 38Â°C prior to use for insemination. Four to six hours after insemination, the eggs were washed and cultured in potassium simplex optimized medium (Quinn & Begley, 1984). PolyA RNA was isolated by using mRNA purification kit (Invitrogen, Carlsbad, CA; cat# 610.06) RNA quality analysis and size-selection (>200 tn) were performed on the Bioanalyzer RNA Pico Chip (Agilent Technologies, Santa Clara, CA). RNA-Seq libraries were constructed using the mRNA-Seq Sample Preparation Kit (Illumina, San Diego, CA)
Experimental Factor: cell type
oocyte
Experimental Factor: developmental stage
oocyte GV