Blue:RNA-Seq Experiments:12192013
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Human Nuclei C1 Experiment: 12-19-2013
Purpose
- To optimize human nuclei capture efficiency
FACS Sort
- DAPI/NeuN (APC) stained human nuclei from Yun (J. Chun Lab):
- FACS sort on the Aria II (SCRM)
- Nuclei were maintained on ice
- Added 50ul of sort buffer containing 0.5% FAF-BSA to 1.5ml eppendorf tube for collection
- Gated nuclei on DAPI positive and NeuN Positive
- Sorted ~50,000 DAPI+/NeuN+ nuclei into collection tube (final volume of ~1000ul)
- Maintained on ice. Centrifuged 2000rpm at 4C for 10 minutes. Removed supernatant down to ~10ul. Resuspended pellet with ~20ul 0.5%BSA/PBS using p200 by pipetting about 10 times.
- Counted nuclei using BioRad automatic cell counter: 1000 nuclei/ul
- DAPI/NeuN (APC) stained human nuclei from Yun (J. Chun Lab):
File:12-19-13 hCtx 1568 NeuNpos for C1.pdf
Buoyancy Tests
- Tested buoyancy of nuclei after adding different amounts of C1 Cell Suspension RGT. Note: after incubating several minutes at RT, want to see the nuclei at all planes of focus within well, not congregated at top or bottom of the solution. Tested different rations:
- 8ul nuclei:2ul C1 Suspension
- 7ul nuclei:3ul C1 Suspension
- 6ul nuclei:4ul C1 Suspension (standard used in protocol)
- 5ul nuclei:5ul C1 Suspension
- 4ul nuclei:6ul C1 Suspension
- 3ul nuclei:7ul C1 Suspension
- Noticed that at 8:2 most nuclei were at the bottom; 7:3 showed nice distribution throughout all focal planes; 6:4 and further dilutions showed no nuclei - were at the top of the solution - too buoyant.
C1 Loading
- Run 1: Medium Chip
- Chip primed as per protocol
- Nuclei Prepared:
- 1ul Nuclei (1000)
- 2.5ul 0.5%BSA sort buffer
- 1.5ul C1 Cell Suspension RGT
- Run 1: Medium Chip
- Run 2: Small Chip
- Chip primed as per protocol
- Nuclei Prepared:
- 1ul Nuclei (1000)
- 2.5ul 0.5%BSA sort buffer
- 1.5ul C1 Cell Suspension RGT
- Run 2: Small Chip
- Run 3: Medium Chip
- Chip primed using Blocking buffer containing BSA (resuspended one full aliquot (180ul) of C1 Blocking RGT into 1ng BSA for ~0.5% final)
- Nuclei Prepared:
- 1ul Nuclei (1000)
- 2.5ul 0.5%BSA sort buffer
- 1.5ul C1 Cell Suspension RGT
- Run 3: Medium Chip
C1 Capture Rates
- Each capture site was viewed using a regular inverted microscope (Run 1) or confocal microscope (runs 2-3).
- Capture Table:
' | ' | 7/30/2013 | 9/23/2013 | *12/2/2013 | 12/5/2013 | 12/19/2013 Medium (7:3) | 12/19/2013 Small (7:3) | 12/19/2013 Medium (7:3 & BSA Block) |
[Nuclei/ul] | 120 | 250 | 1400 | 1200 | 1000 | 1000 | 1000 | |
Total Loaded | 360 | 750 | 2000 | 1500 | 1000 | 1000 | 1000 | |
Total Sites Captured | 26 | 33 | 87 | 77 | 68 | 90 | 91 | |
Single | 16 | 21 | 32 | 45 | 56 | 42 | 75 | |
Double | 6 | 23 | 10 | |||||
Multiple | 10 | 12 | 55 | 32 | 6 | 6 | 6 | |
*Mixed with 1000 mESCs |
C1 Run
- ERCC stock (1:100 dilution aliquot from original stock) was diluted 1:20 in C1 loading buffer for final of 1:2000 dilution added to lysis buffer
- Used run 3 chip for RT/PCR reaction
- Time from FACS sort finished at 2pm to running C1 = ~4hours
Results
- We have defined the SOP for nuclei preparation and C1 capture:
- Staining Nuclei with DAPI or PI and NeuN - Sorting DAPI+NeuN+
- Adding 0.5%BSA to sort tube to prevent sticking
- Resuspending sorted nuclei in BSA sort buffer
- Pre-blocking the chip with BSA to prevent sticking
- Preparing nuclei in a 7:3 ratio with C1 suspension RGT for loading
- Loading 1000 nuclei for a medium chip
- We have defined the SOP for nuclei preparation and C1 capture: