GSM9554843: 102325_ago_mir465_2cell_26; Mus musculus; RNA-Seq
For sperm RNA and embryo isolations for single embryo RNA-seq see Trigg and Conine et al. 2024 Small RNA sequencing from sperm was performed largely as previously described10. Briefly, total sperm RNA was isolated and size-selected for small RNAs. In contrast to the original protocol (18-40 nucleotides), libraries in this study were size-selected for 18-24 nucleotide fragments to enrich specifically for mature miRNAs. Libraries were prepared using a modified Illumina Tru-Seq small RNA library prep protocol and sequenced on an Illumina NextSeq 1000/2000 platform. Libraries were mapped and normalized to reads per million as described previously (Trigg and Conine 2024).  Single-embryo RNA-sequencing. Following microinjection, individual embryos were collected 28 hours (late 2-cell), 46 hours (4-cell), or 70 hours (compacted morula) post activation or fertilization in 5 μL of TCL buffer (Qiagen, Cat. No. 1070498) with 1% 2-mercaptoethanol. Samples were immediately placed in -80°C until all replicates were collected. Libraries were generated using the SMART-Seq protocol11. Briefly, lysed embryos were isolated using RNAClean-XP beads and polyadenylated RNA was reverse transcribed using Superscript II. cDNA was amplified and indexed using the Nextera XT kit and sequenced on an Illumina NextSeq 1000/2000 platform (paired-end 50 bp, ~5 million reads per sample). Reads were mapped against the Mus musculus (mm10) genome using RSEM.