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## Example Calculation of cDNA Yield |
Concentration: 2005.93 pg/μl Elution volume:40 μl; Dilution Factor:10 |
## Total Yield |
= Conc'n (pg/μl) x Elution Vol. (μl) x Dilution Factor 1000 (pg/ng) |
=2005.93 x 40 x 10 1000 (pg/ng) |
= 802.37 ng |
Carrying Forward ONLY 25% of total cDNA yield for GEX Library |
= Total cDNA x 0.25 = 802.37 ng x 0.25 = 200.59 ng |
Refer to step 3.5 for appropriate number of Sample Index PCR cycles based on carry forward cDNA product yield. |
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## Step 3: |
## 3' Gene Expression Library Construction |
| 3.0 Get Started | 51 | |
|------------------------------------------------------------------------------|------| |
| Step Overview (Step 3.1d) | 52 | |
| 3.1 GEX Fragmentation, End Repair & A-tailing | 53 | |
| 3.2 GEX Post Fragmentation, End Repair & A-tailing Double Sided - SPRIselect | 55 | |
| 3.3 GEX Adaptor Ligation | 56 | |
| 3.4 GEX Post Ligation Cleanup - SPRIselect | 57 | |
| 3.5 GEX Sample Index PCR | 58 | |
| 3.6 Post Sample Index PCR Double Sided Size Selection - SPRIselect | 59 | |
| 3.7 Post Library Construction QC | 60 | |
3 |
## 3.0 Get Started |
| Item | Item | 10x PN | Preparation & Handling | Storage | | |
|---------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------|--------------------------------------------------|-----------|--------| |
| Equilibrate to Room Temperature | Equilibrate to Room Temperature | Equilibrate to Room Temperature | | | | |
| □ | Fragmentation Buffer | 2000091 | Thaw, vortex, verify no precipitate, centrifuge. | -20°C | | |
| □ | Ligation Mix | 2001109 | Thaw, vortex, verify no precipitate, centrifuge. | -20°C | | |
| □ | Dual Index Plate TT Set A Verify name & PN. Use indicated plate only | 3000431 | - | -20°C | | |
| □ | Beckman Coulter SPRIselect Reagent | - | Manufacturer's recommendations. | - | | |
| □ | Agilent Bioanalyzer High Sensitivity Kit If used for QC | - | Manufacturer's recommendations. | - | | |
| □ | DNA High Sensitivity Reagent Kit If LabChip used for QC | - | Manufacturer's recommendations. | - | | |
| □ | Agilent TapeStation ScreenTape & Reagents If used for QC | - | Manufacturer's recommendations. | - | | |
| Place on Ice | Place on Ice | Place on Ice | | | | |
| □ | Fragmentation Enzyme Ensure that Fragmentation Buffer and Fragmentation Enzyme from the same kit are used together. Lots are matched for optimal performance | 2000090 /2000104 | Centrifuge briefly. | -20°C | | |
| □ | DNA Ligase | 220110/220131 | Centrifuge briefly. | -20°C | | |
| □ | Library Amp Mix or Amp Mix | 2000531 or 2000047/2000103 | Vortex, centrifuge briefly. | -20°C | | |
| □ | KAPA Library Quantification Kit for Illumina Platforms | - | Manufacturer's recommendations. | - | | |
| Obtain | Obtain | Obtain | Obtain | Obtain | Obtain | |
| □ | 10x Magnetic Separator B | 2001212 | See Tips & Best Practices. | Ambient | | |
| □ | Qiagen Buffer EB | - | Manufacturer's recommendations. | Ambient | | |
| □ | Prepare 80% Ethanol Prepare 15 ml for 8 reactions. | - | Prepare fresh. | Ambient | | |
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## Step Overview (Step 3.1d) |
## Correlation between input & library complexity |
A Single Cell Gene Expression library is generated using a fixed proportion (10 μl, 25%) of the total cDNA (40 μl) obtained at step 2.3m. The complexity of this library will be comparable to one generated using a higher proportion (>25%) of the cDNA. The remaining proportion (30 μl, 75%) of the cDNA may be stored at... |
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Note that irrespective of the total cDNA yield (ng), which may vary based on cell type, targeted cell recovery etc., this protocol has been optimized for a broad range of input mass (ng), as shown in the example below. The total number of SI PCR cycles (step 3.5d) should be optimized based on carrying forward a fixed p... |
## Example: Library Construction Input Mass & SI PCR Cycles |
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