GSM6894165: 2Cell, control, RNA-seq, rep13; Mus musculus; RNA-Seq
2Cell embryos were washed with PBS and collected in lysis buffer without extraction of mRNA Individual two-cell embryos were collected in a 1x TCL buffer (Qiagen) containing 1% beta-mercaptoethanol (Sigma). Total RNA were purified by 2.2x SPRI beads (Beckman) and the beads were resuspended in 6 µL of lysis buffer. Samples were incubated at 72 °C for 10 min and on ice afterwards. Next, 2 µL of reverse transcription mix were added to each sample. Reverse transcription and template switching were carried out at 42 °C for 90 min, then 10 cycles of 50 °C for 2 min, and 42 °C for 2 min, and 85 °C for 5 min. PCR preamplification was performed by adding 12 µL of PCR mix. PCR was conducted as follows: 3 min at 98 °C, 19 cycles of 20 s at 98 °C, 30 s at 65 °C, 6 min at 72 °C, and 5 min at 72 °C. PCR product was purified by 0.8x SPRI beads and eluted in nuclease-free water. Purified cDNA was quality checked by a bioanalyzer and quantified using Qubit 1X dsDNA HS Assay Kit (Thermo Fisher Scientific). Sequence libraries were prepared using Illumina Nextera XT DNA Library Prep kit (Illumina). Libraries were sequenced at paired end (45 bp for read 1 and 30 bp for read 2) 30-bp on an Illumina NextSeq500 instrument.