Three biological replicate ChIP‐seq experiments were conducted for the specific detection of AML1‐bound genomic regions on THAP10 and miR‐383 promoters according to standard procedures with several modifications. Briefly, cross‐linked chromatin from approximately 5–10 × 107 SKNO‐1, SKNO‐1‐siA/E, U937 and U937‐A/E cells was prepared and fragmented to an average size of approximately 200 bp by 30–40 cycles of sonication (30 s each) in 15 ml tubes using the Bioruptor UCD‐200 sonicator (Diagenode). For immunoprecipitation, AML1 (sc‐8563, Santa Cruz Biotechnology), ETO (sc‐9737, Santa Cruz Biotechnology), HDAC1 (ab7028, Abcam), DNMT1 (ab13537, Abcam), DNMT3a (ab13888, Abcam), DNMT3b (ab13604, Abcam) and p300 (ab14984, Abcam) antibodies were added to 12 ml of diluted, fragmented chromatin. Nonimmunized rabbit serum served as a control. ChIP using the normal mouse IgG (Abcam) antibody was performed on naked and sonicated DNA extracted from the same cell samples and assayed using the EZ‐ChIP™ Chromatin Immunoprecipitation kit (Millipore) as per the manufacturer's instructions. Genomic THAP10 and pre‐miR‐383 upstream regions close to the putative AML1‐binding site were amplified. Primer sequences are shown in Appendix Table S7 . GAPDH served as a control for nonspecific precipitated sequences.
Bioruptor ucd 200 sonicator
Manufactured by Diagenode
Sourced in Belgium, United States
The Bioruptor UCD-200 is a benchtop ultrasonic sonicator designed for the efficient disruption of biological samples. It utilizes ultrasonic waves to mechanically shear and homogenize a wide range of sample types, including cells, tissues, and other biomolecules. The device provides consistent and reproducible results by allowing users to precisely control the sonication time and power settings.
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Lab products found in correlation
Qiaquick pcr purification kit, by Qiagen (3 mentions)
Ab7028, by Abcam (2 mentions)
Ab13537, by Abcam (2 mentions)
Ab13604, by Abcam (2 mentions)
Ab14984, by Abcam (2 mentions)
Ab13888, by Abcam (2 mentions)
High sensitivity chip kit, by Abcam (2 mentions)
Ab23980, by Abcam (2 mentions)
Normal mouse igg, by Abcam (1 mentions)
Ez chip chromatin immunoprecipitation kit, by Merck Group (1 mentions)
H3k4me3, by Merck Group (1 mentions)
Nextera dna library preparation kit, by Illumina (1 mentions)
Minelute pcr purification kit, by Qiagen (1 mentions)
Truseq chip sample preparation kit, by Illumina (1 mentions)
H3k4me2, by Merck Group (1 mentions)
H3k9 14ac, by Diagenode (1 mentions)
H3k27ac, by Abcam (1 mentions)
Anti myod, by Santa Cruz Biotechnology (1 mentions)
Sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
15 protocols using bioruptor ucd 200 sonicator
1
Profiling AML1 Genomic Occupancy
2
Chromatin Profiling by ChIP-seq and ATAC-seq
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ChIP and ChIP-seq were performed as previously described (30 (link),45–46 (link)). Briefly, cells were harvested and fixed with 1% formaldehyde. Chromatin was fragmented using a Bioruptor™ UCD200 sonicator (Diagenode SA, Belgium) on high power at 4°C and precipitated using antibodies against CTCF (Millipore, #07-729), H3K4me3 (Millipore, #04-745), H3K4me2 (Millipore, 07-030), H3K27Ac (Abcam, #ab4729) and H3K9/14ac (Diagenode, #C15410005). ChIP-DNA was recovered using phenol/chloroform/isoamyl alcohol and assayed by quantitative polymerase chain reaction (qPCR). ChIP primers are listed in the Supplementary Table S1 . For ChIP-seq, the ChIP DNA library was prepared by ‘TruSeq ChIP Sample Preparation Kit’ (Illumina, #IP-202-1024). Assay for transposase-accessible chromatin using sequencing (ATAC-seq) was performed as previously described (30 (link)). Briefly, 5 × 104 cells were harvested and lysed in lysis buffer on ice. Nuclei were extracted and processed by ‘Nextera DNA Library Preparation Kit’ (Illumina). Libraries were purified with the Qiagen MinElute PCR Purification Kit. ChIP-seq and ATAC-seq libraries were sequenced using the Hiseq2500 (100PE) platform with 25M reads per sample.
3
ChIP-qPCR Analysis of MyoD and H3K27me3
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Chromatin was cross-linked for 12 min with 1% formaldehyde (Sigma) and glycine was then added to a final concentration of 0.125 M for 5 min. After washing and collecting the cells, samples were incubated in nuclei lysis buffer (50 mM Tris-HCl pH 8.1, 1% SDS, 10 mM EDTA) on ice for 30 min. Sonication to obtain chromatin fragments of around 200–300 bp was performed using a Bioruptor UCD-200 sonicator (Diagenode). Chromatin extracts were immunoprecipitated overnight on a rotating platform at 4 °C with 3 μg the following antibodies: anti-MyoD (Santa Cruz, sc-760) and anti-trimethyl-histone H3 (Lys27) (Millipore 07-449). Normal rabbit IgG was used as negative control (mock). Immunoprecipitated chromatin was conjugated with G-protein magnetic Beads (Invitrogen). After extensive washing, bound DNA fragments were eluted and analysed by quantitative PCR using the SYBR Green Master Mix (Applied Biosystems). Primers sequences are indicated in Supplementary Table 2 .
4
Epigenetic Profiling of Chondrocytes
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Primary chondrocytes were isolated from patients with CS and the controls, and cross-linked chromatin was prepared, which was then broken up into fragments of approximately 200 bp in size using a Bioruptor UCD-200 sonicator (Diagenode). These fragmented chromatin samples were incubated with specific antibodies (anti-H3K4me3 antibody, 1:200 dilution, cat no. ab8580, Abcam; anti- H3K9me3 antibody, 1: 200 dilution, cat no. ab8898, Abcam; anti- H3K27me3 antibody, 1: 100 dilution, cat no. ab6002, Abcam; anti- H3K36me3 antibody, 1: 200 dilution, cat no. ab9050, Abcam; anti- H3K79me3 antibody, 1: 200 dilution, cat no. ab2621, Abcam) overnight at 4 °C. Rabbit serum was used as a control. RNA elution and purification were performed using the High-Sensitivity ChIP Kit (ab185913, Abcam) according to the manufacturer’s instructions. The KAT6B promoter was detected by qPCR.
5
ChIP Assay Protocol for RUNX1 and DNMTs
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ChIP assays were performed using the Simple ChIP® Plus Enzymatic Chromatin IP Kit (9005#; Cell Signaling Technology, Danvers, MA, USA) according to the manufacturer’s instructions. Briefly, cross-linked chromatin from approximately 4 × 108 SKNO-1 cells or 4 × 108 SKNO-siAE cells was prepared and fragmented to an average size of approximately 200 bp by 35 cycles of sonication (30 s each) in 1.5 mL tubes using a Bioruptor UCD-200 sonicator (Diagenode, Sparta, NJ, USA). After sonication, chromatin was immunoprecipitated overnight with 5 µg of antibodies against RUNX1 (ab23980; Abcam), RUNX1T1 (ab195329; Abcam), DNMT3A (ab2850; Abcam), DNMT3B (ab2851; Abcam), or DNMT1 (ab92314; Abcam). Normal mouse immunoglobulin G (IgG) served as a negative control. DNA fragments obtained without antibody were used as the input controls. The UBXN8 genomic upstream region surrounding the RUNX1-binding site and unrelated negative control region were amplified by SYBR Green qRT-PCR analysis (Takara) on an Mx3000P LightCycler (Stratagene). The primer sequences are shown in Additional file 1 . Table of primers. The expression was analyzed by the 2−ΔΔCt method and reported as the value relative to that of the input controls. GAPDH served as an internal control accounting for nonspecific precipitated sequences.
6
STAT3 Binding at CSF3R 5'UTR
7
MYCN Chromatin Immunoprecipitation Protocol
8
Whole-Genome Sequencing Library Prep
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A total of 2 µg genomic DNA (35 ng/ml) was fragmented into 300 bp using the Bioruptor UCD-200 sonicator (Diagenode, Denville, NJ) for each sample. Library preparation was constructed using the Kapa Hyper DNA library prep kit for the Illumina platform. Fragmented DNA was end-repaired with an end-repair enzyme and a deoxyadenosine was added to the 3’ ends of the fragments. Kapa barcoded DNA and Kapa indexed adapters were ligated to the sample libraries. The adapter-ligated libraries were selected for an average insert size of 300 bp using next-generation sequencing cleanup and size selection kit (NucleoMag, Macherey–Nagel, Duren, Germany) according to the manufacturer’s protocols. The quality of libraries was assessed using the Bioanalyzer 4200 (Agilent Technologies, Santa Clara, CA). The libraries were then quantified by qRT-PCR and then sequenced were performed by Illumina Nova-seq platform to generate 150-bp paired-end reads.
9
Exome Sequencing of KN9204 Genome
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Genomic DNA was quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Approximately 2 μg of genomic DNA was fragmented into 200–300 bp using the Bioruptor UCD-200 sonicator (Diagenode, Denville, NJ). Libraries were constructed using the DNA library prep kit for the BGI platform (BGI, Shenzhen, China). Fragmented DNA was end-repaired with an end-repair enzyme and deoxyadenosine. According to the manufacturer’s instructions, the adapter-ligated libraries were selected for an average insert size of ∼350 bp using magnetic beads (Twist Bioscience, CA, USA). The pre-capture library amplification was performed with four or five PCR cycles. Equal amounts of products from eight libraries were pooled to obtain 4 μg DNA for hybridization.
Hybridization of sample libraries was performed using the customer exome panel (Tcuni, Chengdu, China). The exome panel was designed specifically for the coding sequences of the KN9204 genome and included 1 232 636 probes covering 127 Mb of the genome (98.11% of the coding sequences of high-confidence genes). Hybrids were captured using the M1 capture beads kit (Thermo Fisher, MA, USA), washed, and amplified by ligation-mediated PCR. The quality of the captured libraries was assessed using the Agilent 4200 Tape Station (Agilent, CA, USA). The final captured libraries were sequenced with the BGI T7 platform.
Hybridization of sample libraries was performed using the customer exome panel (Tcuni, Chengdu, China). The exome panel was designed specifically for the coding sequences of the KN9204 genome and included 1 232 636 probes covering 127 Mb of the genome (98.11% of the coding sequences of high-confidence genes). Hybrids were captured using the M1 capture beads kit (Thermo Fisher, MA, USA), washed, and amplified by ligation-mediated PCR. The quality of the captured libraries was assessed using the Agilent 4200 Tape Station (Agilent, CA, USA). The final captured libraries were sequenced with the BGI T7 platform.
10
AML1-ETO Binding Site Identification on SETDB2 Promoter
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To detect the AML1-ETO binding region on the SETDB2 promoter, a ChIP-seq experiment was performed. First, after knocking down AML1-ETO in Kasumi-1 cells, cells were harvested, and cross-linked chromatin was prepared, which was then broken up into fragments of about 200 bp in size using a Bioruptor UCD-200 sonicator (Diagenode). These fragmented chromatins was incubated with specific antibodies ((AML1 (ab23980, Abcam), ETO (ab124269, Abcam), HDAC1 (ab7028, Abcam), HDAC3a (ab13537, Abcam), HDAC3b (ab13888, Abcam), HDAC3c (ab13604, Abcam), p300 (ab14984, Abcam) overnight at 4 degrees. Rabbit serum was used as a control. RNA elution and purification were performed using the High-Sensitivity ChIP Kit (ab185913, Abcam) according to the manufacturer’s instructions. The expression of the sub-AML1 binding region in SETDB2 promoter was detected by qPCR. GAPDH was used as a control.
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