title: scRNA-seq of homo sapiens: peripheral blood mononuclear cells: Sample DFCI-5341-T-02
design: Cryopreserved samples were thawed the day of experiments. Dead cells were removed via an OptiPrep selection protocol such that cells collected just below the phosphate-buffered saline (PBS) layer were >95% viable on trypan blue staining. Viable cells were subjected to immunomagnetic selection (MACS CD19 MicroBeads; Miltenyi Biotec, catalog no. 130-050-301) using MS columns to isolate CD19+ and CD19− cells. Cells were prepared at a concentration of 1.15 × 105 cells/ml in a 15% OptiPrep solution in PBS and kept on ice until time of encapsulation. Cell encapsulation was performed in a light-protected environment using an Olympus CKX53 microscope, Point Grey Chameleon31.3 MP Mono USB3 Vision camera, and OEM Syringe Pump Modules. RT/lysis mix and barcoded hydrogel beads (BHBs; from 1CellBio) were prepared as previously described (18) and directly before time of encapsulation. During encapsulation, cells and RT/lysis mix were kept at 4°C in their respective syringes using refrigerated copper coiling, and the collection tube was kept cool on an ice rack. All reagents were simultaneously loaded onto microfluidic devices (microfluidic chip; 1CellBio). The Harvard Apparatus Pump Software and the Point Grey camera software were used to control the parameters of encapsulation with the following working flow rates: 50 to 70 ul/hour for BHBs, 340 to 380 ul/hour for carrier oil, 250 ul/hour for cell suspension, and 250 ul/hour for RT/lysis mix, which produced a bead occupancy of 70 to 85% and droplet sizes ranging from 2.5 to 3.5 nl. Encapsulation time for 3000 cells was approximately 9 to 12 min with a calculated cell doublet percentage of 5.45 to 7.22%. Library preparation and sequencing proceeded in the following manner. Briefly, after the target number of cells was encapsulated, the emulsion of droplets was exposed to ultraviolet (UV) light (365 nm at ~10 mW/cm2, UVP B-100 lamp) for 8 min on ice. The emulsion was incubated at 50°C for 2 hours, 70°C for 15 min, and 4°C for 1 min before splitting into 3000 cell aliquots. The emulsion was broken using 10 ul of 20% (v/v) 1H,1H,2H,2H-perfluorooctanolin in Novec-7500 and stored in −80°C until libraries were ready to be processed. In vitro transcription was performed overnight for 16 hours at 37°C. Fragmentation of amplified RNA libraries was performed for 1.5 min at 70°C. The final sequence-ready libraries were amplified by seven to nine polymerase chain reaction (PCR) cycles on average as determined by a diagnostic quantitative PCR. Libraries were quantified by Agilent BioAnalyzer and multiplexed with unique PCR indices in sequencing batches of up to 12,000 cells. The NextSeq Illumina Sequencer was used to sequence libraries using custom sequencing primers.
attributes.tag: alignment_software
attributes.value: tophat2