GSM2550986: S12D067; Mus musculus; RNA-Seq
Embryos were collected in the M15 medium. One single embryo was used at a time, which was incubated in EDTA-PBS, and then treated by Trypsin. The parietal endoderm and ectoplacental cone were easily removed, and the remaining embryo was transferred into the MEM medium with 10% FBS before dissociating into single-cell suspension. The single cells were picked into a lysate buffer by a glass pipette assembled on a micromanipulator, followed by reverse transcription directly on the whole-cell lysate. For cells from E5.5 and E6.5_Early embryos (I-III), and cells from E6.5_Late embryo (X), cDNAs were prepared according to previous methods. Briefly, oligo (dT) primers, which were complementary to poly (A) tails of mRNAs, were incorporated into the 5' end of the first-strand cDNAs during the reverse transcription step. And then a poly (A) tail was added to the 3' end of the first-strand cDNAs by terminal deoxynucleotidyl transferase. After the second strands were synthesized, cDNAs were amplified by 20+12 cycles of PCR. For cells from embryos E6.5_Late (VII) and E6.5_Late (VIII), mRNAs were reverse transcribed by SMARTScribeTM Reverse Transcriptase and then directly amplified by 18+12 cycles. Amplified cDNAs were first examined for the expression of a few marker genes (Oct4, Hand1, Gata6 or a combination of germ-layer markers) by quantitative real-time PCR. cDNA from selected cells were purified either by gel electrophoresis or Agencourt AMPure XP beads. For samples purified by gel electrophoresis, only transcripts with length > 500 bp were cut from the gel. For samples purified with Agencourt AMPure XP beads, 80% ethonal was used so that the transcripts recovered are mainly >500bp. cDNA libraries were prepared for sequencing using standard Illumina protocols
GEO Accession
GSM2550986