title: Illumina HiSeq 4000 paired end sequencing; scRNA-seq of mESC and mouse CD4+ cells using different buffers and enzymes
library_construction_protocol: Cells were collected from cultured mouse ESC cell line. FACS was used to enrich the cells. When FACS sorting, take care of plate calibration/priming prior to single-cell deposition. If many plates are deposited at the same time, repeat the calibration/priming at least every 8 plates. Defrost lysis buffer plates prior to cells sorting, centrifuge (1,000g for 1 min at 4oC) and keep chilled on ice. After FACS sorting, centrifuge immediately (1,000g for 1 min at 4 oC) the plate containing cells and keep them chilled on ice or at -80 oC until further processing.  Transfer 1.2 μl of normalized samples by the Zephyr instrument to the working Nextera Plate. Keep plates chilled and continue with the Nextera protocol.  Prepare the Nextera master mix.  Once the temperature reaches 4 oC, remove from the thermal cycler and centrifuge (1,000g for 1 min at 4 oC) the plate.  Wash Mantis LV chip with water by dispensing 50-100 μl of water into the waste plate.  Dispense 1.2 μl NT buffer into each well to neutralize the reaction.  Nextera Index preparation Timing 30min Prepare the Indexes set mix (A-B-C-D) in 96-well Index Plate according to the manufacturer's instructions. Defrost and spin down the Index 1(i7) and Index 2 (i5). Arrange the index primers in the Index Plate Fixture as follows: Index 1 (i7) adapters in columns 1-12 in ascending or descending order. Index 2 (i7) adapters in columns A-H in ascending or descending order.    Using a multichannel pipette, add 10 μl of i7 adapters down each row. Replace the vial cap with the new cap. Using the multichannel pipette, add 10 μl of i5 adapters down each column. Replace the vial cap with the new cap.  Seal the plate and centrifuge (1,000g for 1 min at 4 oC), vortex at 1800g for 1min and centrifuge (1,000g for 1 min at 4 oC) again.  Keep premade plates with index barcodes chilled until use or in the freezer at -20 oC for long term storage.  Dispense 2.5 μl of Index Adapters on the Zephyr instrument to the Nextera plate from step 55. Seal the plate and centrifuge (1,000g for 1 min at 4 oC).  Nextera Libraries amplification Timing 50min Wash Mantis LV chip by dispensing 50-100 μl of water into the waste plate.  Dispense 3.7 μl of NPM buffer by the Mantis dispenser to each well of the Nextera plate.  Seal the plate and centrifuge (1,000g for 1 min at 4 oC), vortex at 1800g for 1min and centrifuge (1,000g for 1 min at 4 oC) again.  Once the temperature reaches 4 oC, remove from the thermal cycler. Centrifuge the plate (1,000g for 1 min at 4 oC), vortex at 1800g for 1min and centrifuge (1,000g for 1 min at 4 oC) again.