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== Human Nuclei C1 Experiment: 12-02-2013 == == Purpose == *To generate single nuclei RNA-seq libraries using the C1 system on nuclei prepared from the human brain *To "Spike-in" B6 mESCs for control library preparation == FACS Sort == :*DAPI stained human nuclei from Yun (J. Chun Lab): :**Added ~1ul DAPI just prior to FACS sort on the Aria II (SCRM) :**Nuclei were maintained on ice :**Added ~50ul of sort buffer containing 0.5% FAF-BSA to low-bind 1.5ml eppendorf tube for collection :**Gated nuclei on DAPI positive and shifted gate slightly along the FSC axis to exclude the smallest nuclei :**Sorted 150,000 nuclei into the collection tube (final volume of ~1200-1500ul) :**Maintained on ice. Centrifuged 2000rpm at 4C for 10 minutes. Removed supernatant down to ~50ul. Resuspended pellet (not visible) using p1000 by pipetting about 5 times. :**Counted nuclei using BioRad automatic cell counter: '''1480 nuclei/ul''' :*B6 mESCs from Dilyana Todorova (Y. Xu Lab): :**mESCs were collected using 0.25% Trypsin, washed with PBS and stained using SSEA1-FITC (1ul in 100ul PBS) for 20 minutes. DAPI was added just prior to sort. :**mESC were gated on high FITC and no DAPI staining and 100,000 sorted into an eppendorf tube containing ~50ul PBS :**Cells were centrifuged at 1000 rpm for 5 minutes and supernatant removed down to ~50ul. Cell pellet was resuspended using p1000 :**Cells were counted using BioRad cell counter: '''715 cells/ul''' == C1 Loading == :*Medium Chip was primed :*Nuclei and cells were mixed in a 1:1 ratio (7.5ul huNu/7.5ul mESC/10ul C1 cell suspension RGT) for the following total number loaded: :**~2000 huNu :**~1000 mESCs :*Note: these numbers were chosen based on the fact that the previous C1 loading of ~750 huNu only resulted in 33 capture sites and that the mESC expected size was ~9um which would require 2000-3000 for efficient loading. Therefore we used a little over twice the number of nuclei and "spiked-in" mESCs at a sub-optimal loading quantity that would be expected to achieve a low number of capture sites. == C1 Capture == :*Unfortunately the confocal could not be used, so we viewed each capture site using a regular inverted microscope. This was time consuming and the identity of mESCs versus huNu was ambiguous in several cases (note that the DAPI added to the huNu after sort may also have stained the mESCs upon mixing of the two). Several capture sites showed clustering of the huNu (10+) indicating their sticky tendency within the chip. :*Capture Table: {| {{table}} | align="center" style="background:#f0f0f0;"|'''Capture Site''' | align="center" style="background:#f0f0f0;"|'''# of Nuclei''' | align="center" style="background:#f0f0f0;"|'''# of mESCs''' |- | 1||1||0 |- | 2||1||0 |- | 3||1||? |- | 4||0||1 |- | 5||4||1 |- | 6||1||? |- | 7||10||0 |- | 8||2||0 |- | 9||3-4||0 |- | 10||5||0 |- | 11||1||0 |- | 12||20+||0 |- | 13||4||1 |- | 14||1||0 |- | 15||1||1 |- | 16||7||0 |- | 17||2||0 |- | 18||1||0 |- | 19||0||1 |- | 20||5||1 |- | 21||1||1 |- | 22||8+||0 |- | 23||2||1 |- | 24||1||0 |- | 25||2+1F||1 |- | 26||9+||0 |- | 27||6+||0 |- | 28||2||1 |- | 29||3||1 |- | 30||3||0 |- | 31||2||2 |- | 32||2||0 |- | 33||10||0 |- | 34||3||1 |- | 35||1||1 |- | 36||2||2 |- | 37||1||2 |- | 38||1||0 |- | 39||10+||0 |- | 40||4||0 |- | 41||2||1 |- | 42||5F||0 |- | 43||5||1 |- | 44||1||1 |- | 45||1||0 |- | 46||10+||0 |- | 47||10+||0 |- | 48||10+||0 |- | 49||2-3||0 |- | 50||2||0 |- | 51||0||1 |- | 52||2||0 |- | 53||6||0 |- | 54||0||1 |- | 55||1||1 |- | 56||6||0 |- | 57||7||0 |- | 58||2||1 |- | 59||0||1 |- | 60||1||0 |- | 61||1||1 |- | 62||2||0 |- | 63||2||0 |- | 64||5||1 |- | 65||6||0 |- | 66||0||1 |- | 67||1||1 |- | 68||1||? |- | 69||2||2 |- | 70||0||1 |- | 71||1||0 |- | 72||1||1 |- | 73||1||1 |- | 74||1||1 |- | 75||1||? |- | 76||1||0 |- | 77||1||0 |- | 78||5||1 |- | 79||2||1 |- | 80||2||1 |- | 81||1||0 |- | 82||1||0 |- | 83||0||2 |- | 84||1||1 |- | 85||4||0 |- | 86||1||1 |- | 87||2||0 |- | 88||0||1 |- | 89||1||1 |- | 90||5||0 |- | 91||2||1 |- | 92||2-3||0 |- | 93||2||0 |- | 94||3||0 |- | 95||2-3||0 |- | 96||2||0 |- | |} == 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 :*Note that in the RT buffer I may have added the C1 loading buffer twice (only 0.6ul of the 16ul total - so unlikely to affect the reaction efficiency) :*Note that the C1 system's clock was inaccurately set to an hour ahead so that the chip sat with the machine for just over an hour before I was able to collect the samples from it - did not appear as if the samples evaporated any because of this. == Results == :*We were able to achieve a significant enrichment of the nuclei after sorting - this might have been due to the use of BSA in the collection buffer - the nuclei have a tendency of sticking to the plastic tube, therefore the BSA might have prevented this from occurring during the centrifugation step :*Would BSA addition to the chip help prevent nuclei sticking within the chamber sites?
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