Materials and Reagents

Pipette tips 10 μL, 200 μL, and 1,250 μL (BEAVER, catalog numbers: 43140, 43143, 43152)
Pipettes set (Gilson, catalog numbers: GFAM00140)
Centrifuge tubes 15 mL and 50 mL (BEAVER, catalog numbers: 43008, 43009)
EP tube 1.5 mL (AXYGEN, catalog numbers: MCT-150-C-S)
Cell culture plate 6-well, 48-well, 24-well, and 96-well (CORNING, catalog numbers: 3516, 3548, 3526, 3599)
100 mm cell culture plates (BEAVER, catalog numbers: 43701)
HNSC cell line SAS (RIKEN BRC, catalog numbers: RCB1974)
Human embryonic kidney cell line HEK293T (ATCC, catalog number: CRL-3216)
Bovine growth serum (HyClone, catalog number: SH30541.03)
Dulbecco’s modified Eagle’s medium, DMEM (Gibco, catalog number: 31966047)
Streptomycin-penicillin (Gibco, catalog number: 15140122)
Trypsin-EDTA (Gibco, catalog number: 25300096)
Lipofectamine 2000 (Invitrogen, catalog number: 11668019)
Opti-MEM (Gibco, catalog number: 51985026)
EJ2GFP-puro (Addgene, catalog number: 44025)
pQCXIH-I-SceI (a gift from Dr. John P. Murnane, University of California San Francisco)
Puromycin (Sigma-Aldrich, catalog number: P8833)
Hygrovetine (Selleck, catalog number: S2908)
Round-bottom tubes with cell strainer cap, 5 mL (Falcon, catalog number: 38030)
LY2157299 (SelleckChem, catalog number: S2230)
Olaparib (LC Laboratories, catalog number: O-9201)
PBS (see Recipes)
SAS/HEK 293T medium (see Recipes)

Equipment

Cell culture incubator (Thermo Fisher Scientific, catalog number: 51026283)
Neubauer counting chamber (Carl Roth, catalog number: PC72.1)
Centrifuge (Eppendorf, catalog numbers: 5804)
CytoFLEX flow cytometer (Beckman Coulter, catalog numbers: B49008AC)

Software

FlowJo software for FACS analysis

Procedure

Stable integration of EJ2GFP-puro into SAS cells
1. Twenty-four hours prior to transfection, plate SAS cells in a 6-well plate at 5 × 10⁵ cells per well. Each well contains 2 mL of SAS medium.
2. For each well, prepare one Mix A and one Mix B, each in a 1.5 mL tube. Mix gently with finger flicking and incubate for 5 min.
  Component Mix A Mix B
  EJ2GFP-puro 2 μg -
  Lipofectamine 2000 - 5 μL
  Opti-MEM To 125 μL 120 μL
  Total 125 μL 125 μL
3. Add Mix A to Mix B, mix gently, and incubate at room temperature for at least 20 min.
4. After washing the cells twice with 2 mL of 1× PBS, aspirate the PBS.
5. After adding the mixture to SAS cells, incubate at 37 °C and 5% CO₂.
6. After 6 h of incubation, replace the mixture with SAS medium containing 2 mg/mL of puromycin.
7. Change the SAS medium containing 2 mg/mL of puromycin every 2–3 days until the cells reach 80%–90% confluence.
8. Trypsinize cells with trypsin-EDTA and count them.
9. Mix 60 cells with fresh SAS medium (containing 2 mg/mL of puromycin) to form a 6 mL cell suspension.
10. Add 100 μL of cell suspension to each well of a 96-well plate (100 μL of PBS for wells at the edge).
11. Four hours after plating, record the well with only one cell for subsequent screening.
12. When confluence exceeds 50% of well area, seed cells progressively into 48-well plates, 24-well plates, and 6-well plates. Select the cell line in the best condition for amplification and subsequent experiments. As there is a possibility that the selected monoclonal cell line is puromycin resistant and not EJ2GFP-puro successfully transfected, other monoclonal EJ2-puro-SAS cells will also need to be frozen for reserve.

Generation of I-SceI-induced DSBs
1. Retrovirus preparation
  1. One day before packaging, plate 5 × 10⁶ HEK 293T cells on a 100 mm cell culture plate containing 10 mL of HEK293T medium.
  2. Prepare one Solution A and one Solution B, each in a 1.5 mL tube. Mix gently and incubate for 5 min.
    Component Solution A Solution B
    pQCXIH-I-SceI 20 μg -
    Lipofectamine 2000 - 75 μL
    Opti-MEM To 1,250 μL 1,125 μL
    Total 1,250 μL 1,250 μL
  3. Mix Solution A and Solution B together and let it sit at room temperature for at least 20 min.
  4. Wash the HEK 293T twice with 10 mL of 1× PBS.
  5. Add the mixture (Solution A and Solution B) to HEK 293T and incubate for 6 h at 37 °C and 5% CO₂.
  6. After 6 h of incubation, remove the transfection medium and replace with fresh HEK293T medium. Continue to incubate at 37 °C and 5% CO₂.
  7. After 72 h, harvest virus-containing medium. Virus-containing medium can be stored temporarily at 4 °C for 3 days or in a -80 °C refrigerator for approximately 6 months.
2. Generation of DSBs using I-SceI in SAS cells
  1. One day before infection, plate monoclonal EJ2-puro-SAS in a 6-well plate (containing 2 mL of SAS medium) at 5 × 10⁵ cells per well.
  2. Incubate the monoclonal reporter cells with a mixture of 1 mL of virus-containing medium and 1 mL of SAS medium for 24 h.
  3. After 24 h, select infected cells with fresh SAS medium containing 400 mg/mL hygromycin for 10 days. Refresh the medium every 2–3 days. GFP-expressing cells are clearly visible under fluorescent microscopy (Figure 2).
Figure 2. Representative images of GFP-expressing cells under fluorescent microscopy. Images of SAS (A) without or (B) with I-Scel transfection. Scale bar = 10 µm.
Flow cytometry detection of GFP⁺ cells
1. Wash the infected cells (at least 1 × 10⁶ cells) twice with 1× PBS and trypsinize into single cell suspension.
2. Wash again with 1× PBS, spin at 500 × g for 5 min, resuspend in 1 mL of 1× PBS, and force cells through a flow tube with cell strainer cap.
3. Set the gate of GFP/FITC-Area on flow cytometry to detect the proportion of GFP⁺ cells (Figure 3). The efficiency of Alt-EJ detection by flow cytometry is further validated by two classical inhibitors. One is TGFBR1 inhibitor LY2157299, which leads to an upregulation of Alt-EJ levels. The other is PARP1 inhibitor Olaparib, which leads to a down-regulation of Alt-EJ levels. As shown in Figure 3E, our improved reporter assay accurately reflected the effect of both inhibitors on the regulation of Alt-EJ levels.
Figure 3. Flow cytometry detection of GFP⁺ SAS cells. (A) Negative control without I-SceI expression. (B) Control with I-SceI expression. (C) TGFBRI inhibition with LY2157299 (0.4 μmol/L for 48 h) to increase Alt-EJ. (D) PARP1 inhibition with Olaparib (10 μmol/L for 48 h) to decrease Alt-EJ. (E) Percentage of GFP-positive cells of SAS-EJ2-puro. NC: negative control; *P < 0.05; ***P < 0.001.

Recipes

1× PBS
Dissolve 137 mM of NaCl, 2.7 mM of KCl, 7.5 mM of Na₂HPO₄·12H₂O, and 9.1 mM of KH₂PO₄ in 1 L ddH₂O (pH 7.4).
SAS/HEK 293T medium
445 mL of DMEM supplemented with 50 mL of bovine growth serum and 5 mL of streptomycin-penicillin.

Data analysis

Flow cytometry

Gate cells of interest by setting up plots for forward scatter versus side scatter.
Using the forward scatter height versus forward scatter area excludes the doublets.
Untransfected cells were used as a negative control to identify the gate for detecting GFP expression negative cells. This gate was then used to evaluate the level of GFP expression in the target cells after transfection.

Acknowledgments

This study was supported by the Natural Science Foundation of China (82073007; Q. L.). The authors wish to acknowledge Prof. Jeremy Stark, Prof. Mary Helen Barcellos-Hoff, Prof. John Murnane, and Mr. Trevor Jones for their kind help and support with the results of this study. The protocol described here is adapted from previous studies (Gunn et al., 2012; Muraki et al., 2013).

Competing interests

The authors declare no competing interests.

References

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