title:  Bovine corpus luteum cyclic- Cyclic 2
design: Field of study: Reproductive physiology, bovine female reproductive system species: Bos taurus Animal breed: Holstein  age: 18-20 months heifers (bovine female that has not had any previous pregnancy)  tissue= corpus luteum (transient gland in the ovary, that secretes progesterone which is essential for successful pregnancy) Period= day 17 of the estrous cycle or of pregnancy Keyword search: Corpus luteum, microRNA, cattle, high-throughput sequencing, maternal recognition of pregnancy. Experimental design: 8 heifers were used in this experiment: 4 were not pregnant, and not bred and 4 were bred and confirmed pregnant. on day 17 of the estrous cycle (if non pregnant) or of pregnancy the copora lutea (plural of corpus luteum) were collected and immediately frozen for total RNA extraction (including small RNA). (we used Ambion miRVana kit).  Small RNA were shipped to Beijing Genomic Institute (BGI; Hong Kong, China) for small RNA transcriptome sequencing analysis. Single-end sequencing with a read length of 50 nt was performed on an Illumina HiSeq 2000 sequencer.    Briefly, 18-30 nt length small RNA underwent 3’-adapter ligation followed by 5’-adapter ligation to obtain a 62-75nt long sequence.  A reverse transcription primer, complementary to 3’-adaptor was used for reverse transcription.  After PCR amplification, the purified PCR products were sequenced by HiSeq 2000 sequencer (BGI protocol, Hafner et al., 2008).    Using Illumina base-calling algorithm, each base was assigned a quality score ranging from 0 (low) to 41 (high).  Low quality reads (>4 bases with a quality score < 10) were eliminated from total reads.  The resulting clean reads were analyzed for length distribution to determine the composition of small RNA in each of the 8 samples.    Counts of reads for each length category were normalized for each sample as follows: (number of reads for each length category/total reads) x 100.  Uniformity of sequencing was assessed through the comparison of the percentages of common and sample-specific sequences in all 8 samples, two samples at a time (i.e. total of 28 pairwise comparisons).  Samples with high count of common tags and low count of specific tags indicated a good uniformity of sequencing (BGI protocol,).  