Blue:RNA-Seq Experiments:12192013: Difference between revisions

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:*Each capture site was viewed using a regular inverted microscope (Run 1) or confocal microscope (runs 2-3).   
:*Each capture site was viewed using a regular inverted microscope (Run 1) or confocal microscope (runs 2-3).   


[[File:12-19-13_huNu_C1_74.jpg]]
[[File:12-19-13_huNu_C1_74.jpg|400px]]


:*Capture Table:
:*Capture Table:

Revision as of 00:05, 21 December 2013

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

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 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 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

C1 Capture Rates

  • Each capture site was viewed using a regular inverted microscope (Run 1) or confocal microscope (runs 2-3).

File:12-19-13 huNu C1 74.jpg

  • 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 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