GSM1518966: tagged-908_S4; Mus musculus; RNA-Seq
Mouse embryonic Stem cells (V6.5) and mouse embryonic fibroblast cells (10.5 p.c.) are cultured from cell-lines. The cells are dissociated prior to encapsulation using trypsin and re-suspended in PBS at a concentration of about 5•106/mL. Human derived K562 cells are cultured from cell-line in suspension and re-suspended prior to encapsulation in PBS at a concentration of about 5•106/mL. The cell suspension is loaded in a syringe together with the magnetic stirrer bar that prevents sedimentation of the cell suspension. The cell suspension is injected into a co-flow drop-maker device (Figure 3B) with a 2X lysing buffer containing 100 mM Tris-HCl, 300 mM NaCl and 1 % Triton X-100 and dispersed in oil with 0.2 % w/w surfactant. We use OEM syringe pumps (KD Scientific, MA, USA) with typical flow rates of 1.8 mL/hr for the oil and 250 µL/hr for each of the aqueous phases, resulting in a drop diameter of ~50 µm. Drops are collected and incubated off chip at 4ºC for 15 minutes to complete cell lysis. The lysing protocol was optimized to maximize product after amplification and lysis is verified visually after 15 min. To label cells in drops, cell-bearing drops and barcode-library drops are re-injected into a “three point merger” device. Drops are spaced on chip by oil with 0.2% w/w surfactant and are then electrocoalesced. An additional RT buffer is pico-injected as the drops are coalesced. The buffer contains 1X Affinity Script buffer (Agilent), 10 mM DTT, 8 mM dNTPs, Rnase inhibitor (Rnasin, Promega) diluted 1:10 and Affinity Script Multi Temp RT enzyme (Agilent) diluted 1.5:10. The device electrodes are connected to a high voltage amplifier (TREK 2210) which supplies a 100 V sine wave at a frequency of 25 kHz. The flow rates used to inject the drops are chosen to ensure that no more than one barcode drop fuses with a single cell-bearing drop, even at the expense of some drops not fusing with other drops. The flow rate of the buffer containing RT-enzyme is chosen to ensure that the buffer is added at ~1:1 ratio upon coalescence of the two drops. Typical flow rates fulfilling these requirements are 1 mL/hr for the oil, 100 µL/hr for the cell-bearing drops, 30 µL/hr for the barcode-drops and 170 µL/hr for the RT-enzyme buffer. To control the number of cells collected per sample, we measure the filling number λ at the cell encapsulation stage and use a fast camera (HiSpec1, Fastec Imaging,USA) to measure the frequency f of pairs of barcode-drops and cell-bearing drops that fuse at the labeling stage. The time T100 required to collect 100 cells is then calculated as T100= 100/λf. Typically, f=100 Hz and λ=0.1, so that 1000 pairs of fused drops are collected to sample 100 cells, and the collection lasts 10 seconds. To protect the collected drops from evaporating and adsorbing to the walls of the collection vial, they are collected into a vial containing ~50 µL of oil and 1 % w/w surfactant and 30 µL of emulsion of ~70 µm carrier drops containing 0.25X Affinity Script buffer, 25 mM Tris-HCl, 75 mM NaCl and 0.25 % Triton X-100. The sample is collected and incubated at 42 ºC for 1 hour to allow the RT reaction to occur; thereafter it is maintained at 4 ºC. Barcodes were commercially synthesized (IDT) and delivered in three 384 well-plates at concentrations of 150 µM. To encapsulate the barcodes in drops we design 96 parallel drop-makers on a single microfluidic chip (Figure 3A), so that the aqueous inlets of each drop-maker (22 gauge stainless steel capillaries, New England Small Tube) precisely fit one quarter of a 384 well-plate and are immersed in 96 different wells, each containing a unique barcode. Oil with 1% w/w surfactant is distributed to all drop-makers via a common inlet that is connected to a pressurized oil reservoir. The plate and the microfluidic parallel device are placed in a pressure chamber while a common outlet for all 96 barcode drop-makers is located outside the pressure chamber. Upon pressurizing the chamber, each of the 96 barcode solutions is forced through its own drop-maker, thereby forming an emulsion where every drop contains about 1 billion copies of one of the 96 barcodes. We pressurize the oil reservoir to 9 psi and the pressure chamber to 6 psi, producing ~35 µm drops at a rate of about 500 µL/min from 96 wells. After encapsulation the device is washed with water by placing it in a petri dish filled with water and pressurizing the chamber for 2 minutes. Then the device is similarly washed with isopropanol with the exception that the oil inlet is also fed with isopropanol. Finally, the device is dried by placing it in the pressurized chamber with all inlets exposed for several minutes. The process is repeated 12 times in 6 hours until a total of 1152 different barcodes are encapsulated to form the final barcode-library emulsion. A total volume of about 10 mL is produced from 10 µL in each well. RT preformed within the microfluidic drops. The RT primers includes polyT barcode and illumina anchor. after breaking the drops we followed with RNAse treatment and ligated a ss-DNA adapter containg a second illumina anchor to the cDNA 3' end. Size selected for 100bp-500bp
GEO
GSM1518966
GEO Accession
GSM1518966