12/24/2023 0 Comments 10x chromium single cell protocolLoad Row Labeled 1 with 70uL Master Mix without introducing bubbles. DO NOT ADD WATER DIRECTLY INTO THE CELL SUSPENSION. Gently pipette to mix the cells suspension before adding to the Master Mix. Add the appropriate volume of nuclease free water to the Master Mix, then add the corresponding volume of cell suspension to the Master Mix, for a total of 75 µl in each tube. Refer to the Cell Suspension Volume Calculator Table. Important: Do not pipette anything into the NO FILL wells at the bottom row of the chip 45 µl to unused wells in row labeled 3.50 µl to unused wells in row labeled 2.70 µl to unused wells in row labeled 1.If loading less than 8 samples on one chip, pipette 50% glycerol into the unused wells without introducing bubbles. Each column of wells on the chip is occupied by one sample.Close the lid of the holder over the chip. Gently press down the right side of the chip until it clicks into place. The chip label should be at the top and facing toward you. Keep these tubes on ice.Īssemble the Next GEM Chip G by inserting it in the Secondary Holder under the guide (on the left side of the holder when open). Prepare Master Mix on ice, per sample (10% extra has already been included):Īdd 31.8 uL Master Mix into each tube of a PCR 8-tube strip. If NOT using a new kit, retrieve Template Switch Oligo (already resuspended in TE Buffer) from -80C storage and allow to equilibrate to room temp for at least 30 minutes. If using a new kit: resuspend Template Switch Oligo in 80uL Low TE Buffer and leave at room temp for at least 30 minutes. Thaw RT Reagent B and Reducing Agent B at room temp. 10X Genomics recommends 700-1,200 cells/µl in order to maximize cell encapsulation while avoiding excessive multiplets.įor more detail on cell preparation, refer to 10X’s literature, which covers:Īllow the Single Cell 3’ v3.1 Gel Beads to come to room temperature for 30 minutes before loading the chip. Since each bead has a single barcode, transcripts from multiplets will appear as though they are from the same cell. Multiplets occur when a single oil droplet contains 1 barcoded bead and two or more cells. While a high concentration of cells is desirable, it also increases the rate of ‘multiplets’. Refer to the Cell Suspension Volume Calculator Table at the end of this section. Depending on your concentration, you will need 2.1-23.6 uL of cell suspension as input for each sample. The optimal input cell concentration is 700-1,200 cells/µl. Cell Suspension (See following Instructions).Single Index Kit T Set A (-20C Freezer).Chromium Next GEM Chip G Single Cell Kit (Room Temp). Chromium Next GEM Single Cell 3ʹ GEM, Library & Gel Bead Kit v3.1 (2 boxes in -20C Freezer and 1 box in -80C Freezer).Earlier versions of kits/reagents will not be compatible 10X reagents (included): Important: Our machine is only compatible with v3.1 Next GEM reagents.
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