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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''' [[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|400px]] :*Capture Table: {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''12/19/2013 (Run 1) Medium (7:3)''' | align="center" style="background:#f0f0f0;"|'''12/19/2013 (Run 2) Small (7:3)''' | align="center" style="background:#f0f0f0;"|'''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 == :*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
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