GSM2672061: C12.8 (RNA-Seq); Homo sapiens; RNA-Seq
Single cells were isolated from blastocyst stage embryos (6-7 days post fertilisation) for subsequent analysis by micromanipulation, with a duration of less than 20 minutes. Embryos were placed in drops of G-MOPS solution (Vitrolife) on a petri dish overlaid with mineral oil. The plate was placed on a microscope stage (Olympus IX70) and the embryos were held with an opposing holding pipette and blastomere biopsy pipette (Research Instruments) using Narishige micromanipulators (Narishige, Japan). The biopsy mode of a Saturn 5 laser (Research Instruments) was used to separate the majority of the mural TE from the ICM and polar TE. The ICM and polar TE were washed quickly in PBS without Ca2+ and Mg2+ (Invitrogen) then placed in 0.05% trypsin/EDTA (Invitrogen) for 5 minutes at room temperature. Trypsin was quenched using Global Media supplemented with 5 mg/mL LifeGlobal Protein Supplement. After quenching, the cell clump was placed back on the stage in a drop of G-MOPS solution and pipetted up and down several times using the blastomere biopsy pipette.Single cells were picked using 100 μm inner diameter Stripper pipette (Origio) and transferred to individual low bind RNAse-free tubes containing 2.5 µl RLP plus buffer. Samples were processed using a previously published protocol that was adapted where indicated (Macaulay et al. 2016).To separate RNA from gDNA 50 µl of Dynabeads were washed and incubated with 100 uM biotinylated poly-dT oligonucleotide (IDT). 10 µl of oligo-dT beads were added to each tube containing the single cell. Samples were incubated in a thermomixer for 20 min at room temperature at 2000 rpm. Tubes were put on a magnet until the beads collected into a pellet and the supernatant went clear. The supernatant containing the gDNA was transferred to a new collection tube. Beads were washed three times to collect any residual gDNA.cDNA was generated from the RNA captured on the bead using the SMARTer v4 Ultra Low Input kit. First strand cDNA was synthesised by adding 2 µl 5X Ultra low First strand buffer, 0.5 ul SMART-Seq v4 Oligo, 0.25 µl RNase Inhibitor, 6.25 µl Nuclease free water and 1 µl SMARTScribe Reverse Transcriptase to each sample. Reverse transcription was performed on the thermomixer using the settings 2 min at 42oC at 2000 rpm,  60 min at 42oC at 1500 rpm, 30 min at 50oCat 1500 rpm and 10 min at 60oCat 1500 rpm. cDNA was amplified by adding 12.5 μl 2X SeqAmp PCR buffer, 0.5 μl  PCR Primer II A (12µM), 0.5 µl SeqAmp DNA polymerase, 1.5 μl Nuclease free water. Beads were mixed on thermomixer for 60 sec at room temperature at 2000 rpm and then were incubated on a PCR machine using the following settings: 95oC for 1 min, 24 cycles of 98oC for 10 sec, 65oC for 30 sec and 68oC for 3 min, before a final extension for 10 min at 72oC. Amplified cDNA was purified by adding 25 μl SPRI Ampure XP beads, mixing well and incubating at room temperature for 8 min to allow amplified cDNA to bind to the beads. Sample tubes were placed on the magnet and allowed to stand until all beads had been immobilised. Supernatant was removed and discarded and beads were washed twice by adding 200 µl freshly prepared 80% ethanol and leaving for 30 sec before discarding the supernatant. Tubes were spun briefly to collect residual liquid. The bead pellet was allowed to air dry. 12 µl of purification buffer was added to rehydrate the pellet and incubated for 2 min at room temperature. cDNA was eluted by pipetting up and down 10 times before returning the tube to the magnet. The clear supernatant containing the cDNA was removed from the immobilised beads and transferred to a new low-bind tube. cDNA was stored at -80°C until library preparation. cDNA quality was assessed by High Sensitivity DNA assay on an Agilent 2100 Bioanalyser with good quality cDNA showing a broad peak from 300 to 9000 bp. cDNA concentration was measured using QuBit dsDNA HS kit (Life Technologies UK Ltd.).Libraries with a molar concentration greater than 2nM were submitted for 100-bp paired-end sequencing on Illumina HiSeq 2000.