GSM1624235: TE8.3; 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. cDNA was generated from single cells using the SMARTer Ultra Low Input RNA kit for Illumina Sequencing?HV (Clontech Laboratories, Inc.) according to maunfacturers? guidelines. Single cells were picked using 100 ?m inner diameter Stripper pipette (Origio) and transferred to individual low bind RNAse-free tube containing 0.25 ?l RNase inhibitor, 4.75 ?l Dilution buffer and 5 ?l nuclease-free water on a -80°C pre-chilled CoolRack (Biocision, CA). Samples were stored at -80°C until ready to be processed. 1 ?l of 3? SMART CDS Primer II A was added to the sample, mixed well and incubated at 72°C for 3 min. First strand cDNA was synthesised by adding 4 ?l 5X First-Strand Buffer, 0.5 ?l 100 mM DTT, 1 ?l 20 mM dNTP mix, 1 ?l SMARTer IIA Oligonucleotide, 0.5 ?l RNase Inhibitor and 2 ?l SMARTScribe Reverse Transcriptase (100 U/?l) directly to a tube containing the sample and incubating at 42°C for 2 hours followed by 10 min at 70°C. First strand cDNA was purified by adding 36 ?l of room temperature SPRI Ampure XP beads (Beckman Coulter Genomics), mixing well and incubating at room temperature for 8 min. Tubes were placed on a MagnaBot II Magnetic Separation device (Promega) and allowed to stand until all beads were immobilised into a pellet. The supernatant was removed and discarded. Tubes were briefly spun and any residual liquid was removed. Double stranded cDNA was amplified from the template bound to the beads using Advantage 2 PCR kit (Clontech Laboratories, Inc.). 5 ?l 10X Advantage 2 PCR Buffer, 2 ?l 10 mM dNTP Mix, 2 ?l IS PCR Primer, 2 ?l 50X Advantage 2 Polymerase Mix and 39 ?l Nuclease-Free water were added to the tube containing the sample to give a total volume of 50 ?l. PCR amplification was performed at 95°C for 1 min, followed by 18 cycles of 15 sec at 95°C, 30 sec at 65°C and 6 min at 68°C followed by final extension step of 10 min at 72°C. Amplified cDNA was purified by adding 90 ?l SPRI Ampure XP beads, mixing well and incubating at room temperature for 8 min to allow amplified cDNA 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 ?l 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 ?l 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.) Typical yields from a single cell ranged from 1 ng to 9 ng. In preparation for library generation cDNA was sheared using Covaris S2 to achieve cDNA in 200-500 bp range. 10 ?l of cDNA sample and 65 ?l purification buffer was added to Covaris AFA Fiber Pre-Slit Snap Cap microTUBE. cDNA was sheared using the settings 10% Duty, Intensity 5, Burst Cycle 200 for 2 min. Sheared cDNA was transferred to a new 0.2 ml low-bind tube. Libraries were prepared using Low Input Library Prep Kit (Clontech Laboratories, Inc.) according to manufacturer?s instructions. The amount of input cDNA was calculated from the concentration measured by the Bioanalyser assay prior to shearing, taking into account the dilution involved in the shearing step. The appropriate amplification cycle number was selected according to manufacturer?s guidelines. Library quality was assessed by Bioanalyser and the concentration was measured by QuBit assay.  The molar concentration of library was calculated thus: Library molecular weight = average size in bp (from Bioanalyser) x 650 g/mol per bp.