5 protocols
nucleic acid sequencing protocol
The small RNA molecules were ligated a pair of adaptors (5'adaptor-GTTCAGAGTTCTACAGTCCG-ACGATC, 3' adaptor-TCGTATGCCGTCTTCTGCTTG) sequentially. Then the small RNAs were reverse transcription by reverse-transcription polymerase chain reaction (RT-PCR) amplifications. Finally, the purified RT-PCR products were used for sequencing by the Illumina Genome Analyzer (Illumina, San Diego, CA, USA) according to the manufacturer's instructions.
nucleic acid library construction protocol
Library preparation and sequencing was performed at BGI-Shenzhen.
treatment protocol
All the five patients were stimulated with the standard long gonadotrophin-releasing hormone agonist (GnRHa, Diphereline; Ipsen Pharma. Biotech, Signes, France) protocol combined with the administration of recombinant FSH (Gonal-f, Merk Serono SA, Geneva, Switzerland). For oocyte retrieve, all patients underwent ovarian puncture (OPU) of follicles larger than 15 mm after 36 h of administration of 10 000 IU human chorionic gonadotrophin (hCG, LiZhu Pharma, ZhuHai, China). Only the cumulus'oocyte complexs with Metaphase II (MII) oocytes were included in this study.
nucleic acid extraction protocol
Total RNAs were extracted from the human CRCs and COCs using TRIzol reagents (Invitrogen).
growth protocol
Briefly, the cumulus' oocyte complexes were retrieved 36 h after hCG treatment and washed in multiple dishes with Flushing medium (William A. Cook Australia Pty. Ltd, Queensland, Australia). The cumulus oophorus cells were collected in the fertilization medium (William A. Cook Australia Pty. Ltd, Queensland, Australia) by two disposable needles and two 1-ml plastic disposable syringes without hyaluronidase. To avoid the interfusion of corona radiata cells, the innermost layers of cumulus oophorus cells were not collected. On the other hand, the corona radiata cells were collected as described in our previous publication and other reports. The corona radiata cells separated from the oocyte by gentle pipetting with a 135 mm diameter stripper pipette and micromanipulator System. MII oocytes were used for ICSI procedure. The corona radiata cells and cumulus oophorus cells were pooled by centrifuging at 1500g for 8 min, and immediately frozen in liquid nitrogen until use