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

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| align="center" style="background:#f0f0f0;"|'''7/30/2013'''
| align="center" style="background:#f0f0f0;"|'''12/19/2013 (Run 1) Medium (7:3)'''
| align="center" style="background:#f0f0f0;"|'''9/23/2013'''
| align="center" style="background:#f0f0f0;"|'''12/19/2013 (Run 2) Small (7:3)'''
| align="center" style="background:#f0f0f0;"|'''*12/2/2013'''
| align="center" style="background:#f0f0f0;"|'''12/19/2013 (Run 3) Medium (7:3 & BSA Block)'''
| align="center" style="background:#f0f0f0;"|'''12/5/2013'''
| align="center" style="background:#f0f0f0;"|'''12/19/2013 Medium (7:3)'''
| align="center" style="background:#f0f0f0;"|'''12/19/2013 Small (7:3)'''
| align="center" style="background:#f0f0f0;"|'''12/19/2013 Medium (7:3 & BSA Block)'''
|-
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| [Nuclei/ul]||||120||250||1400||1200||1000||1000||1000
| [Nuclei/ul]||||1000||1000||1000
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| Total Loaded||||360||750||2000||1500||1000||1000||1000
| Total Loaded||||1000||1000||1000
|-
|-
| Total Sites Captured||||26||33||87||77||68||90||91
| Total Sites Captured||||68||90||91
|-
|-
| ||Single||16||21||32||45||56||42||75
| ||Single||56||42||75
|-
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| ||Double||||||||||6||23||10
| ||Double||6||23||10
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| ||Multiple (>2)||10||12||55||32||6||6||6
| ||Multiple (>2)||6||6||6
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| ||||||||*Mixed with 1000 mESCs||||||||
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Latest revision as of 00:08, 21 December 2013

Human Nuclei C1 Experiment: 12-19-2013[edit]

Purpose[edit]

  • To optimize human nuclei capture efficiency


FACS Sort[edit]

  • 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[edit]

  • 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[edit]

  • 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[edit]

  • 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:
' ' 12/19/2013 (Run 1) Medium (7:3) 12/19/2013 (Run 2) Small (7:3) 12/19/2013 (Run 3) Medium (7:3 & BSA Block)
[Nuclei/ul] 1000 1000 1000
Total Loaded 1000 1000 1000
Total Sites Captured 68 90 91
Single 56 42 75
Double 6 23 10
Multiple (>2) 6 6 6

C1 Run[edit]

  • 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[edit]

  • 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