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Stanford Genome Technology Center


Barcode quantification by Tag Array

Method Overview

The following steps describe quantification of barcodes using the Genflex Tag 16K Array from Affymetrix, which was specifically designed for compatibility with the Yeast Knock-Out Collection (Pierce et al. 2006). DNA barcodes can also be quantified using next-generation sequencing (Smith et al. 2009; Smith et al. 2010; Gresham et al. 2011; Robinson et al. 2013), and we also include an alternative protocol for this approach.

Protocol

  1. PCR amplify UP and DOWN tags from each genomic DNA sample in separate reactions (see PCR Reaction Mixture recipes ) using the following conditions:

  2. i. 94°C for 3 minutes

    ii. 94°C for 10 seconds
    iii. 55°C for 30 seconds
    iv. 72°C for 30 seconds
    --> go to step ii. 29 times

    v. 72°C for 3 minutes

    vi. 4°C hold


  3. Verify that the PCR reactions produce the expected 60bp product by applying gel electrophoresis to 5 µL of each reaction. PCR products can be stored at -20°C for up to 3 months.
    (Note: A smaller band of 40bp is often observed in both the template and no template reactions. This represents common primers forming a primer dimer and, if of lower intensity than the 60bp band, will not affect the hybridization.)

  4. Hydrate one GenFlex Tag16k array for each sample with 75 µL of 1X Hybridization Buffer (see recipe) , and incubate chips at 42°C at 20 rpm for 10-20 minutes in an Affymetrix Hybridization Oven.

  5. Prepare the Tag Hybridization Mixture (see recipe)for each sample in 0.5 mL Safe-Lock microfuge tubes.

  6. Float each tube in boiling water for 2 minutes, transfer immediately to ice for 2 minutes, spin down condensate, and leave on ice.

  7. Remove 1X Hybridization Buffer from each array, add 90 µL of Tag Hybridization Mixture, and cover both gaskets with Tough-spots.

  8. Incubate arrays in an Affymetrix Hybridization oven for 16 hours at 42°C, rotating at 20 rpm.

  9. Immediately prior to washing the arrays, prepare Chip wash buffer A , Chip wash buffer B , and the labeling mixture (see recipes).

  10. Wash and label microarrays using an Affymetrix Fluidics Station 450 with the protocol FlexGenflex_Sv3_450 with the following modifications: change the temperature of the wash B step from 40°C to 42°C, and change the temperature of the staining step from 25°C to 42°C.

  11. Place Tough-spots over both gaskets, check glass slide for cleanliness and bubbles. Glass can be cleaned with a Kimwipe and isopropanol, and bubbles can be removed by slowly pipetting Chip wash buffer A in and out of the array.

  12. Scan arrays using Affymetrix GeneChip Scanner

Recipes

PCR Reaction Mixture

(Final Concentration of 1X reaction buffer: 1mM MgCl2, 0.08 mM dNTPs, 1 M each UPTAG and BUPKANMX4 oligonucleotides, 0.05 U/L)

Prepare 50 µL ‘UP tag’ PCR Reaction Mix for each sample as follows:

5 µL of 10X reaction buffer
2 µL of 25 mM MgCl2
0.4 µL of 10 mM dNTPs
0.5 µL of 100 µM UPTAG oligonucleotide: (GATGTCCACGAGGTCTCT)
0.5 µL of 100 µM BUPKANMX4 oligonucleotide: (Biotin-GTCGACCTGCAGCGTACG)
0.5 µL of 5 U/µL Taq polymerase
~75 ng of purified genomic DNA
De-ionized water to 50 µL

PCR-amplify the ‘DOWN tags’ in the same way, but use DNTAG oligonucleotide: (CGGTGTCGGTCTCGTAG) and BDNKANMX4 oligonucleotide: (Biotin-GAAAACGAGCTCGAATTCATCG), in place of UPTAG and BUPKANMX4, respectively.

2X Hybridization Buffer

(Final Concentration of 2X Hybridization Buffer: 1X MES, 1.77 M NaCl, 40 mM EDTA, 0.02% Tween 20)

First prepare ~5 mL of 0.66 M MES Hydrate (Sigma M5287) and ~5 mL of 1.78 M MES sodium salt (Sigma M5057). Mix equal volumes of each to create a 12X MES stock solution.

Use this to make 50 mL of 2X Hybridization Buffer as follows:

8.3 mL of 12X MES stock
17.7 mL of 5 M NaCl
4 mL of 500 mM EDTA
100 L of 10% Tween 20
19.9 mL of de-ionized water

This can be stored at 4C for up to 3 months.

Tag Hybridization Mixture

(Final Concentraion of Tag Hybridization Mixture: 1X Hybridization Buffer, 0.66nM B213 oligonucleotide, 1 M each of the 8 blocking oligonucleotides, 1X Denhardts)

Prepare 90.5 L for each sample as follows:

75 L of 2X Hybridization Buffer
0.5 L of 200 nM B213 oligonucleotide (Biotin-CTGAACGGTAGCATCTTGAC)
12 L of 12.5 M of each blocking oligonucleotide

o Uptag (GATGTCCACGAGGTCTCT)
o Dntag (CGGTGTCGGTCTCGTAG)
o Uptagkanmx (GTCGACCTGCAGCGTACG)
o Dntagkanmx (CGAGCTCGAATTCATCG)
o Uptagcomp (AGAGACCTCGTGGACATC)
o Dntagcomp (CTACGAGACCGACACCG)
o Upkancomp (CGTACGCTGCAGGTCGAC)
o Dnkancomp (CGATGAATTCGAGCTCG)

3 L of 50X Denhardts (Sigma D2532)

Add 30 L of UP tag PCR product and 30 L of DOWN tag PCR product for a final volume of 150.5 L.

Chip Wash Buffer A

(Final Concentraion of Chip Wash Buffer A: 6 X SSPE, 0.01%Tween)

Prepare 500 mL as follows and filter sterilize:.

150 mL of 20X SSPE (Invitrogen 15591-043)
500 µL of 10% Tween 20
349.5 mL de-ionized water

 

Chip Wash Buffer B

(Final Concentraion of Chip Wash Buffer B: 3 X SSPE, 0.01%Tween)

Prepare 500 mL as follows and filter sterilize:.

75 mL of 20X SSPE (Invitrogen 15591-043)
500 µL of 10% Tween 20
424.5 mL de-ionized water

References

  1. Pierce SE, Fung EL, Jaramillo DF, Chu AM, Davis RW, Nislow C, Giaever, G. 2006 .A unique and universal molecular barcode array. Nat Methods 8: 601-3. PMID: 16862133.
  2. Smith AM, Durbic T, Kittanakom S, Giaever G, Nislow C. 2012. Barcode sequencing for understanding drug-gene interactions. Methods Mol Biol 910: 55-69. PMID: 22821592
  3. Smith AM, Heisler LE, Mellor J, Kaper F, Thompson MJ, Chee M, Roth FP, Giaever G, Nislow C. 2009. Quantitative phenotyping via deep barcode sequencing. Genome Res 19: 1836-1842. PMCID: PMC2765281
  4. Robinson DG, Chen W, Storey JD, Gresham D. 2013. Design and Analysis of Bar-seq Experiments. G3 (Bethesda). PMCID: PMC3887526
  5. Gresham D, Boer VM, Caudy A, Ziv N, Brandt NJ, Storey JD, Botstein D. 2011. System-level analysis of genes and functions affecting survival during nutrient starvation in Saccharomyces cerevisiae. Genetics 187: 299-317. PMCID: PMC3018308

     



Inquiries can be addressed to Maureen Hillenmeyer (maureenh at stanford.edu) and Angela Chu (amchu at stanford.edu)
Stanford Genome Technology Center