Genotyping HSC-derived colonies by barcoded NGS

Single HSC Genotyping

  1. Sort Single HSC in Methocult in 96 well plate. One HSC per well. After 12-14 days observe colony formation.
  2. Individual colonies can be collected from the plate by adding 200ul DPBS in each well and collect in Eppendorf.
  3. Spin down slowly (1500 rpm x 5 min) and wash cell pellet with 200uL DPBS.
  4. Spin down and resuspend in lysis buffer for DNA isolation.
    1. For each sample, need 50uL Viagen Lysis reagent + 1uL Proteinase K
  5. Place tubes in 56°C water bath for 20 min followed by 95°C incubation for 10 min.
  6. Store DNA long term at –20C.

Perform 2-step PCRs

  1. Use the isolated genomic DNA, setup the first “adaptor PCR” to amplify the target region.
    • Use EconoTaq high-fidelity polymerase and run the program for 25-30 cycles (cycle # and Anneal temp will depend on primer set optimization)
      • Setup 20uL reactions: 10ul 2x EconoTaq MM + 1uL each primer (20uM) + 1-2uL DNA template + NF-H2O up to 20uL
      • PCR program: 94C x 2min -> [94C x 15 sec – 55C x 15sec – 72C x 40 sec] -> 72C x 5 min -> hold 4C
  2. Purify product from adaptor PCR using Sera magnetic beads.
    • Add 1.8x volume of beads per reaction, mix well and incubate @RT for 10mins
    • Place the PCR tubes on magnet and wait for 2 mins and discard the supernatant.
    • Wash twice using 200ul of 80% ethanol and airdry beads for 5mins.
    • Take the tubes from magnetic stand and add 15ul water to the beads for elution and incubate for 10mins.
    • Elute the sample and use the purified sample for second set of PCR.
  3. Setup the 2nd “index PCR” using 2uL purified product from adaptor PCR.
    • Each sample will have its own unique F index primer based on the indexes.
      • All samples will have universal R primer (JM 890)
    • Use EconoTaq polymerase and run program for 12 cycles
      • Setup 20uL reaction: 10uL 2x EconoTaq MM + 7.6uL NF-H2O + 0.2uL each primer (universal paired end and index, 100uM) + 2uL purified PCR-1
      • PCR program: 94C x 1min -> [94C x 30 sec – 60C x 30sec – 72C x 40 sec] -> 72C x 5 min -> hold 4C
  4. Add 2uL 6x loading dye to each sample and load 5ul to the 1.5% agarose gel and check for the right size bands.
  5. Pool all the remaining samples together (different index) and load in a separate gel.
  6. Extract indexed samples from the gel by removing bands in the correct size range using the UV light and a scalpel—transfer each gel slice to a labeled Eppendorf tube
  7. Weigh all the samples as well as an empty tube to determine the mass of each gel slice and calculate the volume of NT1 to be used.
  8. Perform gel purification via Takara cleanup kit, eluting 15uL in water at the last step.
  9. Measure concentrations of indexed samples using the NanoDrop.
Notes

NOTE THAT THE NANODROP VALUES WILL BE OFF SIGNIFICANTLY
The SIC recommends increasing the target concentration by 3-fold to alleviate discrepancies

 

  1. Calculate sample dilutions required to get equal pooling with the final concentration of 1.8ng/ul per sample.
  2. Dilute each sample to 10nM in 20uL H2O and Combine equal volumes of diluted sample to 20uL.
  3. The sample can be stored at –20C at this step if needed or directly submit samples to MiSeq spike-in cooperative (SIC).