Microwell-Seq. High-throughput Single Cell RNA-Seq Kit. Protocol. Index

Transcription

1 Microwell-Seq High-throughput Single Cell RNA-Seq Kit Protocol Index 1 Introduction 2 Kit Reagent 3 Store 4 Application 5 Prepared materials 6 Note 7 Preparation 8 Workflow 1

2 1 Introduction Microwell-Seq High-throughput single cell RNA-Seq Kit (Microwell-Seq Kit). Based on Microwell-seq technology, one stop process from cell suspension to cdna can be realized. Microwell-seq confines single cells and barcoded poly(dt) mrna capture beads in a PDMS array of subnanoliter wells.well dimentions are designed to accommodate only one bead.cells are loaded by gravity with a rate of dual occupancy that can be tuned by adjusting the number of cells and loaded and visualized prior to processing. 2 Kit Reagent The Microwell-Seq Kit consists of Box1 and Box2 kits. Order No.is SC , for two samples. Reagent Volume Quantity BOX1 Capture Beads 110 l 1 Wash Buffer 50 ml 1 SSC Buffer 25 ml 1 prea Buffer 0.6 ml 1 preb Buffer 1 ml 1 TES Buffer 1 ml 1 TET Buffer 1 ml 1 Lysis Buffer 0.6 ml 1 Betaine 10 l 1 MgCl 2 10 l 1 Pure Beads 200 l 1 BOX2 ddh ml 1 RT Buffer 10 l 1 dntp 10 l 1 DTT 10 l 1 TSO primer 5 l 1 Rtase 10 l 1 Rnase Inhibitor 5 l 1 ExoI 25 l 1 ExoI Buffer 50 l 1 3 Store Kapa 110 l 1 Preamp primer 10 l 1 Box1 stores at 4 o C. Box2 stores at -20 o C except TSO primer stores at -80 o C. 2

3 4 Application Suitable for mammalian cells / eukaryotic cells without cell wall less than 25m in diameter, but not for prokaryotic cells. 5 Prepared materials Instruments Consumables Microscope, vortex shaker, meta bath, PCR, centifuge, Qubit 2.0 or Qubit mm petri dish, razor blade, magnetic frame, magnet, magnetic plate, pipettor(1ml 200l 20 l), pipette tips without enzyme, ice box, plastic tweezer, EP tubes without enzyme, PCR tubes without enzyme When wash Capture beads/pure beads, EP tubes should be out of the magnetic frame. But when discard the supernatant, EP tubes should be put on the magnetic frame; All components in this kit have been optimized. Please don t change the reaction system; The operation flow is recommended to be completed at one time. No interruptions are recommended; Reagents, consumables, etc. in workflow meet for one sample. If there are more than one samples, please increase related reagents, consumables, etc., accordingly. Select active cells for the experiment. 6 Note Reagents 80% etanol 7 Preparation 7-1 Reagents Take reagents out of Box1. Put on ice. Operate on a clean bench. Pipette tips and EP tubes should be free of enzyme; Sufficiently mix reagents before use; Take reagents out of Box2. Melt. Put on ice. Take TSO primer out of -80 C. Melt. Put on ice. 3

4 7-2 Samples Count cultured cells or cell suspension of digested tissue. The cell suspensions containing about cells were centrifuged for 5 minutes. The supernatant was then collected in the same EP tube and the supernatant should be removed away as much as possible. Resuspend with 1ml Wash buffer. Put on the magnetic frame. Discard the supernatant. Resuspend with 500l Wash buffer. Put on ice. 8 Workflow Add 1ml Wash buffer, mix and centrifuge 300g for 5 minutes, then remove the supernatant. Repeat this step. Tissue digested cells need to be filtered, but not the cultured cells. Add Wash Buffer to cell suspension to make the final cell concentration 1.0 ~ / ml. Small cells are low density and large cells are high density. So increase concentrations of large cells appropriately. 7-3 Microwell plate Soak the microwell plate in Wash Buffer at room temperature. 7-4 Capture beads Take 50 l Capture beads stock solutions. Put on the magnetic frame. Discard the supernatant. 8-1 Cell collection 4

5 Add prepared cell suspension to the microwell plate. Then cells fall into wells with gravity. Each well can hold only one cell. About 10% wells will be occupied by cells. 1. Wash the microwell plate with Wash Buffer twice. Discard the supernatant before use. 2. Add l prepared cell suspension to the microwell plate to cover the whole plate. Note: The volume of the added cell suspension depends on the size of the microwell plate. The liquid level is slightly below the edge of the microwell plate. 3. Occasionally tap the desktop for 10 minutes to help cells fall into wells. Observe the microwell plate under a microscope. About 10% wells will be occupied by cells. Note:Prolonging the time can increase the rate of falling well. If cells fall not well, you can re-take a microplate and add cells. 4. Discard the redundant cell suspension along the edge of the microwell plate slowly. 5. Add l Wash Buffer to the microwell plate slowly. Discard along the edge slowly. Repeat this step once. Note: Don t wash violently to prevent cells that have fallen into the wells from being washed off. 6. Discard cell suspension with 200 l pipettor as much as possible. 8-2 Add capture beads Add Capture beads to the microwell plate. Capture beads fall into the wells by magnetism. Each well can hold only one Capture bead. All wells in the microwell plate will be occupied by Capture beads. So, about 10% wells have both cells and Capture beads. 1. Put magnet in the center of the magnetic plate. 2. Add l prepared Capture beads suspension to the microwell plate slowly to cover the whole microwell plate. Put on the magnet. Vortex, <500 rpm, 8 min. Observe under a microscope. Note: The liquid level of the added Capture beads suspension was slightly below the edge of the agarose microwell plate. If the vortex time was too long, there would be multi-capture beads in a well. 3. Carefully cut the three sides of the microwell plate off with a razor blade. The remaining side is convenient for tweezers 5

6 to pick up. 4. Add 2.5ml Wash buffer to a new 35mm petri dish. Carefully put the microwell plate into the buffer. Lift the microwell plate up about 15. Shake gently to wash away the excess Capture beads. Observe under a microscope. Note: Don t wash violently to prevent Capture beads that have fallen into the wells from being washed off. Put the petri dish on the magnet when wash. 5. Transfer the microwell plate to a new 35mm petri dish with less liqiud. 8-3 Cell lysis Add lysis buffer to the microwell plate to release RNA. Then RNA will bind to Capture beads through base pairing. 1. Put the magnet on a 4 C ice box. Then place the petri dish with a microwell plate on the magnet. Note:Place the microwell plate in the center of the petri dish and to keep four corners from touching the edge of petri dish. 2. Slowly add 200 l pre-cooled lysis buffer to the microwell plate and incubate on ice for 15 min. Note:If precipitation in Lysis Buffer, mix. 3. Preparations Set 42 C metal bath. Prepare 20l RT Mix. Put on ice. Reagent ddh2o RT Buffer Betaine dntp DTT MgCl 2 TSO primer RTase Rnase inhibitor Total Volume 7.3l 4l 4l 2l 0.5l 0.3l 0.4l 1l 0.5l 20l Add 2.5ml SSC buffer to a new 35mm petri dish. Put on the magnet. 4. After ice incubation, add 1ml SSC Buffer slowly to the microwell plate to wash. 5. Carefully transfer and invert the microwell plate into pre-added 2.5 ml SSC Buffer petri dish by tweezer. Put on 6

7 the magnet. Shake 30s-1min gently to collect Capture beads. Note: Transfer the microwell plate with as little liquid as possible. You can choose to cut off the redundant side of the microwell plate or not, depending on the edge of the microwell plate. After collection of Capture beads, observe the microwell plate under a microscope. If remaining beads is more than 5%, repeat the collection of Capture beads. 6. Place one 1.5ml EP tube on the magnetic frame. Transfer the liquid with Capture beads in the petri dish to one EP tube. Discard the supernatant. Note: When discard the supernatant, EP tube should always be on the magnetic frame to prevent Capture beads from being washed off.when wash the bottom, petri dish should be taken from the magnet to put on the bench. 7. Wash the bottom of petri dish with 1ml SSC Buffer. Transfer the liquid to the same EP tube. Discard the supernatant to collect Capture beads. 8. Resuspend with 500 l SSC Buffer. Centrifuge several seconds. Discard the supernatant. Note: When resuspend Capture beads, EP tubes should be out of the magnetic frame. But when discard the supernatant, EP tubes should be put on the magnetic frame. Similarly hereinafter. 9. Resuspend with 200l prea Buffer. Discard the supernatant. 8-4 Synthesis of the first cdna chain Synthesize the first cdna chain from RNA bound to Capture beads by reverse transcription. 1. Discard the supernatant as much as possible with 20l pipettor. Resuspend with 20l RT mix. 2. RT react in 42 C metal bath, 90min. Flick with fingers every 6 minutes. 3. Preparations Prepare 200l exonuclease mix. Put on ice. Reagent ddh 2O ExoI Buffer ExoI Total Volume 170l 20l 10l 200l 7

8 4. After RT reaction, centrifuge several seconds. Discard supernatant. Total 100l 5. Wash capture beads once with 200l TES Buffer. Then wash once with 200l TET Buffer. 6. Wash Capture beads once with 200l preb Buffer. Centrifuge several seconds and discard the supernatant. 4. After the exonuclease reaction, put EP tube on the magnetic frame and discard the supernatant. 5. Wash capture beads once with 200l TES Buffer. Then wash once with 200l TET Buffer. 6. Wash once with 200l preb Buffer. 8-5 Exonuclease degradation Cut off oligonucleotides which did not bind mrna. 1. Set 37 C metal bath. 2. Resuspend with 200 l exonuclease mix. Incubate at 37 for 60 minutes. Flick with fingers every 10 minutes. 3. Preparations Prepare 100l PCR reaction system. Put on ice. Reagent ddh2o Kapa preamp primer Volume 48l 50l 2l 7. Discard the supernatant with 20 l pipettor as much as possible. 8-6 cdna amplification Synthesize the second cdna strand from the first cdna strand and amplify. 1. Resuspend with 100l PCR reaction system. Then distribute the Capture beads to 4 or 5 tubes on ice. 2. PCR react. At the end of 98 C in the first 1-4 cycles, flick with fingers. The PCR program was as follows: 8

9 Note: Do not disturb pure beads when discarding the supernatant. 7. Add 200l 80% ethanol to the PCR tube. Incubate for 30s and discard the supernatant. Repeat once. Note: Do not disturb pure beads when adding ethanol. 3. Preparations Sufficiently mix Pure beads. And put at room temperature for at least 30min. 4. After the PCR reaction, collect PCR product into one PCR tube. Put on the magnetic frame. Transfer the supernatant to a new PCR tube and record the volume of supernatant as V. 5. Resuspend with 0.7V room temperature Pure beads. Incubate at room temperature, 8min. Note: Mix 10 times or more with pipette. 6. Put PCR tube on magnetic frame for more than 5 minutes to ensure that the Pure beads are completely absorbed by the magnetic frame. Discard the supernatant. 8. Put PCR tube on the magnetic frame for 30 s. Discard all residual ethanol. Open the cap and dry for 3 to 5 minutes in the air. Note: Make sure that beads are just dry and no cracks appear. 9. Take PCR tube from the magnetic frame. Add 15l enzyme free ddh 2 O to cover the pellet and mix. Incubate at room temperature for 2 mins. 10. Put on the magnetic frame for more than 5 minutes to ensure the solution is clear. 11. Transfer the supernatant to a new EP tube. 12. Detect concentrations by Qubit 3.0 or Qubit 2.0. Store at -20 C. 9