SALL4 抗体 (AA 1-220)

• Cell harvest
• • Harvest 500,000 human melanoma cells per antibody to be used at RT. /li>
  • Centrifuge cell solution 3 min at 600 x g at RT.
  • Remove the liquid carefully.
  • Gently resuspend cells in 1 mL Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, Roche Complete Protease Inhibitor EDTA-free) by pipetting and transfer cell solution to a 2 mL microcentrifuge tube.
  • Centrifuge cell solution 3 min at 600 x g at RT and discard the supernatant.
  • Repeat twice for a total of three washes.
  • Resuspend cell pellet in 1 mL Wash Buffer by gently pipetting.
• Concanavalin A beads preparation
• • Prepare one 1.5 mL microcentrifuge tube.
  • Gently resuspend the magnetic Concanavalin A Beads (antibodies-online, ABIN6923139).
  • Pipette 10 µL Con A Beads slurry for each sample into the 1.5 mL microcentrifuge tube.
  • Place the tube on a magnet stand until the fluid is clear. Remove the liquid carefully.
  • Remove the microcentrifuge tube from the magnetic stand.
  • Pipette 1 mL Binding Buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1 mM CaCl₂, 1 mM MnCl₂) into each tube and resuspend ConA beads by gentle pipetting.
  • Spin down the liquid from the lid with a quick pulse in a table-top centrifuge.
  • Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
  • Remove the microcentrifuge tube from the magnetic stand.
  • Repeat twice for a total of three washes.
  • Gently resuspend the ConA Beads in a volume of Binding Buffer corresponding to the original volume of bead slurry, i.e. 10 µL per sample.
• Cell immobilization – binding to Concanavalin A beads
• • Carefully vortex the cell suspension and add 10 µL of the Con A beads in Binding Buffer to the cell suspension for each sample.
  • Close tube tightly and rotate for 10 min at RT.
• Cell permeabilization and primary antibody binding
• • Divide cell suspension into separate 2 mL microcentrifuge tubes, one for each antibody (500,000 cells per sample).
  • Place the microcentrifuge tubes on a magnetic stand until the fluid is clear. Remove the liquid carefully.
  • Remove the microcentrifuge tubes from the magnetic stand.
  • Place each tube at a low angle on the vortex mixer set to a low speed and add 150 µL Digitonin Wash buffer (wash buffer with 0.025% (wt/vol) Digitonin) supplemented with 2 mM EDTA.
  • Gently vortex the microcentrifuge tubes until the beads are resuspended.
  • Add 1.5 µL antibody (anti-SALL4 antibody ABIN6132627, anti-H3K27me3 positive control antibody ABIN6923144, and anti-FLAG tag antibody negative control) to the respective tube, corresponding to a 1:100 dilution.
  • Rotate the microcentrifuge tubes ON at 4 °C.
  • Spin down the liquid and place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
  • Remove the microcentrifuge tubes from the magnetic stand.
  • Resuspend with 1 mL Digitonin Wash Buffer and mix by inversion. If clumping occurs, gently remove the clumps with a 1 ml pipette tip.
  • Repeat once for a total of two washes.
• pAG-MNase Binding
• • Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
  • Remove the microcentrifuge tubes from the magnetic stand.
  • Vortex the sample at low speed and add 3.75 µL 20X CUTANA pAG-MNase for ChIC/CUT&RUN Assays (ABIN6950951) to 0.5X in 150 µL Digitonin Wash Buffer per sample, gently resuspending the beads by pipetting.
  • Rotate the microcentrifuge tubes for 1 h at 4 °C.
  • Spin down the liquid and place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
  • Remove the microcentrifuge tubes from the magnetic stand.
  • Resuspend with 1 mL Digitonin Wash Buffer and mix by inversion. If clumping occurs, gently remove the clumps with a 1 mL pipette tip.
  • Repeat once for a total of two washes.
• MNase digestion and release of pAG-MNase-antibody-chromatin complexes
• • Spin down the liquid from the lid with a quick pulse in a table-top centrifuge.
  • Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
  • Place each tube at a low angle on the vortex mixer set to a low speed and add 100 μL Digitonin Wash buffer per sample along the side of the tube.
  • Place tubes in a heat block, kept on ice, and allow to chill.
  • Add 2 μL 0.1 M CaCl₂ to each sample.
  • Incubate tubes at 0 °C for 30 min.
  • Add 100 μL 2xSTOP buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.05% (wt/vol) Digitonin, 100 μg/mL RNAse A, 50 μg/mL Glycogen).
  • Incubate tubes at 37 °C for 30 min.
  • Place the tubes on a magnet stand until the fluid is clear.
  • Transfer the supernatant containing the pA-MNase-bound digested chromatin fragments to fresh 1.5 mL microcentrifuge tubes.
• DNA extraction
• • Add 2 µL 10% SDS to a final concentration of 0.1% and 2.5 µL Proteinase K (20 mg/mL) to each supernatant.
  • Gently vortex tubes at a low speed of approximately 1,100 rpm.
  • Incubate tubes at 50 °C for 1 h.
  • Add 200 µL PCI to tube.
  • Vortex tubes thoroughly at high speed until the liquid appears milky.
  • Centrifuge tubes in a tabletop centrifuge at 16,000 x g at RT for 5 min.
  • Carefully transfer to upper aqueous phase to a fresh 1.5 mL microcentrifuge tube containing 2 µL glycogen (diluted 1:10 to 2 mg/mL from the 20 mg/mL stock solution).
  • Add 20 µL 3 M NaOAc pH 5.2.
  • Add 400 µL 100% ethanol.
  • Place tubes for at -20 °C ON.
  • Centrifuge tubes in a tabletop centrifuge at 16,000 x g at 4 °C for 5min.
  • Remove the liquid carefully with a pipette.
  • Wash pellet with 1ml 70% ethanol.
  • Centrifuge tubes in a tabletop centrifuge at 16,000 x g at 4 °C for 1 min.
  • Remove the liquid carefully with a pipette.
  • Air-dry the pellet, then dissolve in 30 µL 1 mM Tris-HCl, 0.1 mM EDTA.
• Library preparation and sequencing
• • Prepare libraries using KAPA HyperPrep Kit using KAPA Dual-Indexed adapters according to protocol.
  • Sequence samples on an Illumina NextSeq 500 sequencer, using a NextSeq 500/550 High Output Kit v2.5 (75 Cycles), 36bp PE.
• Bioinformatics
• • Align reads the human genome (hg38) using bowtie78 with settings -X 700 -m1 -v 3. Remove duplicate reads, and sort files using samtools. Filter mapped reads for size, keeping only reads with a fragment size at or below 120 base pairs.
  • Generate bedgraph files using bedtools genomecov.
  • Call peaks using SEACR version 1.3, in relaxed mode, normalizing to the negative control.

Results are published in Diener, J., Baggiolini, A., Pernebrink, M. et al. Epigenetic control of melanoma cell invasiveness by the stem cell factor SALL4. Nat Commun 12, 5056 (2021). https://doi.org/10.1038/s41467-021-25326-8