Illumina HiSeq 2000 paired end sequencing; RNA sequencing of mouse embryonic development
RNA sequencing of mouse embryonic development
Mouse strains were intercrosses between Pdgfra::GFP (Hamilton et al. Mol Cell Biol. 23:4013-4025) and strain 129 estrous-checked females. Embryos were collected from the oviduct or uterus in M2 medium (Sigma) at progressive developmental stages between E2.5 and E5.5. Embryonic diapause was induced by surgical removal of both ovaries from female mice on the morning of the third day of pregnancy (E2.5) and embryos were flushed at E6.5. For morula and early blastocyst stages zona pellucidae were removed using acid Tyrode’s solution (Sigma). Blastocysts from E3.25–4.5 were subjected to immunosurgery as previously described (Solter and Knowles, Proc Natl Acad Sci USA 72:5099-102; Nichols et al. Cell 95:379-91). Briefly, blastocysts were incubated for 15min in a 1:5 dilution of anti-mouse rabbit serum (Sigma) in N2B27 medium, washed in N2B27 and further incubated for 15min in a 1:5 dilution of rat serum (in-house) in N2B27 for the complement reaction. Embryos were transferred into N2B27 without washing and incubated for 15min to allow efficient cell lysis. The ICM was subsequently dissociated from residual trophectoderm with a narrowly fitting glass pipette. For postimplantation embryos the epiblast was isolated by manual dissection. Extraembryonic ectoderm was first removed with a wide mouth-controlled pipette, and the visceral endoderm was then mechanically separated by drawing the embryonic portion through a narrow glass aperture. Dissociation of morulae, ICMs and postimplantation epiblast was performed in a 1:1 mixture of 0.025% trypsin plus EDTA (Invitrogen) and 0.025% trypsin (Invitrogen) plus 1% chick serum (Sigma). Morulae and early ICMs were incubated for 5min at 37 C, and E4.5 ICMs and postimplantation epiblasts were incubated for 10min. The explants were washed in N2B27 supplemented with 10µM HEPES and 1.5mg/ml BSA (Gibco) and dissociated in a small drop of medium using blunted microcapillaries with an inner diameter large enough to accommodate approximately 2–3 cells. Cells of the primitive endoderm lineage were distinguished from epiblast cells by Pdgfra::GFP fluorescence and manually sorted under a dissection microscope. Mouse ES cells were dissociated by conventional methods in bulk culture; single-cell suspensions were prepared and 20 cells were manually selected with a blunted microcapillary for further processing. Cells were transferred immediately into 4.5ul of single-cell lysis buffer and snap frozen on dry ice. cDNA synthesis was performed using whole transcriptome preamplification (Tang et al. Nat Methods 6:377-382, Tang et al. Nat Protoc. 5:516-535). cDNA was preamplified for 20 cycles using a single-temperature protocol in a Veriti 96 Well Thermal Cycler (Applied Biosystems). Preamplified cDNA samples were sheared by ultrasonication on a Covaris S2 for 80s with the following parameter settings: Duty Cycle = 10, Cycles per Burst = 200, Intensity = 4. Samples were ethanol precipitated and resuspended in Tris buffer for subsequent enzymatic modifications. End repair was carried out with T4 DNA polymerase and T4 polynucleotide kinase (New England Biolabs) and samples were purified with Ampure XP paramagnetic beads (Beckman Coulter). Blunt-end, 3'-phosphorylated products were 3'-adenylated with exo- Klenow fragment in the presence of dATPs (New England Biolabs), purified with Ampure beads and ligated to sequencing adapters (Illumina) by T4 DNA ligase at 20 C for 30 minutes. PCR amplification of library constructs was carried out with AccuPrime DNA polymerase (Invitrogen) for 13 cycles on a Bio-Rad C1000 thermocycler using denaturing and annealing conditions optimized for even A/T vs. G/C processing (Aird et al. Genome Biol 12:R18). Molarity and size distribution of sequencing libraries were assessed by DNA 1000 microfluidic chips on the 2100 Bioanalyzer (Agilent).
Experimental Factor: developmental stage
expanded blastocyst
Experimental Factor: organism part
primitive endoderm
LIBRARY_STRAND
not applicable