| description |
The RNA-seq experiment was designed to determine the effects of blastocyst transfer on the transcriptome of whole placentas at embryonic day (E) 10.5. C57Bl/6 blastocysts that were from natural matings without superovulation were transferred into the uteri of pseudopregnant B6D2F1 females (at gestational day 2.5 equivalent). RNA libraries were prepared from whole male placentas at E10.5 (N = 4 placentas) from the transfer group alongside RNA libraries from control placentas from C57Bl/6 conceptuses derived from natural matings and did not undergo the transfer process (N = 4 placentas). Libraries were muliplexed and pooled for sequencing on the Illumina NextSeq500 platform with 75-base-pair single-end reads. Sequencing was performed in duplicate to provide at least 18 million reads per sample, and 1% PhiX Control (Illumina) spike-in was used. Quality control of Fastq files was performed using FastQC and fastq_screen. Sequences were trimmed with Trim Galore!, and alinged to GRCm38 mouse genome using STAR aligner.Alignments were processed using custom ClusterFlow (v0.5dev) pipelines and assessed using MultiQC (0.9.dev0). Gene quantification was determined with HTSeq-Counts (v0.6.1p1). Additional quality control was performed with rRNA and mtRNA counts script, feature counts (v 1.5.0-p2) and qualimap (v2.2). Differential gene expression was performed with DESeq2 package (v1.16.1, R v3.4.0). Read counts were normalised on the estimated size factors. |