Small RNA and transcriptome profiling of mice deficient in either germline Dgcr8 or Dicer

We used Illumina Small RNA and RNA-Seq kits to prepare both small RNA and RNA-Seq libraries from total RNA isolated from either leptotenze/zygotene or pachytene spermatocytes purified from either Dgcr8 or Dicer germline conditional knockout mice. Conditional knockout mice were generated by using a Ddx4 promoter to drive cre excision of either Dgcr8 or Dicer at embryonic day 18. Mixed leptotene/zygotene or pachytene spermatocytes were then isolated from the testis of adult conditional knockout mice, along with paired WT littermates as a control. RNA was isolated from these spermatocytes using Trizol. Small RNA or RNA-Seq libraries were then prepped using Illumina's sequencing library preparation kits.我们通过Ddx4启动子驱动Cre重组酶在胚胎第18天介导Dgcr8或Dicer基因的切除，成功构建Dgcr8或Dicer生殖细胞条件性敲除（germline conditional knockout）小鼠。从上述条件性敲除成年小鼠及其配对的野生型同窝对照的睾丸中，分离得到混合的细线期/偶线期（leptotene/zygotene）或粗线期（pachytene）精母细胞；使用Trizol试剂从上述精母细胞中提取总RNA，随后采用Illumina小RNA及RNA测序（RNA-Seq）建库试剂盒，以所提取的总RNA为材料分别制备小RNA文库与RNA-Seq文库。