ChIP assays were performed as previously described (10 (link)). Experiments were conducted in hormone-depleted MCF-7 cells treated with 0.1% Control EtOH Vehicle or 10nM E2 for 45min. Chromatin was cross-linked with 1% formaldehyde for 10 min at room temperature. Cells were washed with PBS and collected using lysis buffer supplemented with 1× complete protease inhibitor mix (Roche). Chromatin was sonicated in lysis buffer to 300–500 bp size fragments. Antibodies were incubated with cell lysates overnight for chromatin collection and then with Dynabead Protein A and G (Life Technologies) for 6 h. ChIP DNA was isolated using PCR puri cation kit (QIAGEN) per the manufacturer’s instructions and used for quantitative real-time PCR. Amounts of ChIP DNA were normalized to inputs. Antibodies used for ChIP experiments were H3K4me3 (ab8895, Abcam); H3K9me3 (ab8898, Abcam); p53 (DO-1, sc-29435); RNA Polymerase II (29634A, CTD4H8 clone, Upstate); ERα (HC-20, Santa Cruz); ERβ antibodies were a combination of CWK-F12 (produced in our lab) (25 (link)), GTX70182 (GeneTex), GR40 (Calbiochem), and PA1-311 (Af nity Bioreagents); N-CoR (sc-1609, Santa Cruz); and SMRT (sc-1610, Santa Cruz).
Pcr puri cation kit
Manufactured by Qiagen
The PCR purification kit is a laboratory equipment product designed to purify polymerase chain reaction (PCR) products. It is used to remove unwanted components, such as primers, nucleotides, and enzymes, from the PCR reaction mixture, allowing for the isolation of the desired DNA or RNA amplicons.
Automatically generated - may contain errors
Lab products found in correlation
Complete protease inhibitor mix, by Roche (1 mentions)
Dynabead protein a and g, by Thermo Fisher Scientific (1 mentions)
Sc 1610, by Santa Cruz Biotechnology (1 mentions)
Ab8895, by Abcam (1 mentions)
Ab8898, by Abcam (1 mentions)
Erα hc 20, by Santa Cruz Biotechnology (1 mentions)
N cor sc 1609, by Santa Cruz Biotechnology (1 mentions)
Primestar gxl dna polymerase, by Takara Bio (1 mentions)
Nepa21 electroporator, by Nepa Gene (1 mentions)
Genome analyzer 2, by Illumina (1 mentions)
Klenow and dna prime labeling system, by Thermo Fisher Scientific (1 mentions)
Taq master mix, by Vazyme (1 mentions)
Pmd18 t, by Takara Bio (1 mentions)
Epitect bisul te kit, by Qiagen (1 mentions)
6 protocols using pcr puri cation kit
1
ChIP Assay in MCF-7 Cells
2
Hygromycin Resistance Screening in C9 Cells
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A DNA cassette containing the hygromycin (hyg)-resistant gene aphVII driven by the β2-tubulin promoter was ampli ed by PCR from plasmid pHyg3 (Berthold et al., 2002) (link) using PrimeSTAR GXL DNA Polymerase (Takara Bio).
The PCR product was puri ed using a PCR puri cation kit (QIAGEN), and the concentration was adjusted to 100 ng µL - 1 . The gene cassette was used to transform C9 cells by electroporation using a NEPA-21 electroporator (NEPAGENE), as described previously (Yamano et al., 2013) (link). The transformants were incubated at 25˚C for 24 h with gentle agitation under illumination of 1.5 µmol photons m - 2 •s - 1 and then screened on TAP plates containing 30 µg mL - 1 hyg at 25˚C under illumination of 80 µmol photons m - 2 •s - 1 . After 4 days, colonies of transformants appeared and were subjected to the screening process of HC-requiring mutants.
The PCR product was puri ed using a PCR puri cation kit (QIAGEN), and the concentration was adjusted to 100 ng µL - 1 . The gene cassette was used to transform C9 cells by electroporation using a NEPA-21 electroporator (NEPAGENE), as described previously (Yamano et al., 2013) (link). The transformants were incubated at 25˚C for 24 h with gentle agitation under illumination of 1.5 µmol photons m - 2 •s - 1 and then screened on TAP plates containing 30 µg mL - 1 hyg at 25˚C under illumination of 80 µmol photons m - 2 •s - 1 . After 4 days, colonies of transformants appeared and were subjected to the screening process of HC-requiring mutants.
3
Mapping Replication Origins via Nascent-Strand Sequencing
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Replication origins were mapped using the nascent-strand sequencing and abundance assay [22] . Brie y, DNA fractionation was performed using a 5-30% sucrose gradient to collect DNA fractions ranging from 0.5 to 2 kb. Five prime single-strand DNA ends were phosphorylated by T4 polynucleotide kinase (T4PK) (NEB, M0201S). After phenol/chloroform treatment to remove T4 PK, DNA was precipitated, resuspended and then treated with lambda-exonuclease (NEB, M0262S) to remove genomic DNA fragments that lacked the phosphorylated RNA primer. After RNase treatment and DNA puri cation (Qiagen PCR puri cation kit, 28004), single stranded nascent strands were random-primed using the Klenow and DNA Prime Labeling System (Invitrogen, 18187013). Double-stranded nascent DNA (1 μg) was sequenced using the Genome Analyzer II (Illumina).
4
Bisulfite Sequencing for Genomic DNA Analysis
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Traditional bisul te sequencing PCR was used to verify the WGBS data. Each 1 µg sample of genomic DNA was subjected to sodium bisul te using an EpiTect Bisul te kit according to the manufacturer's instructions (Qiagen). The processed DNA was puri ed with a Qiagen PCR puri cation kit (Cat. No. 28106) and used as a template for bisulfate sequencing PCR (BS-PCR). The primers were designed using Kismeth (http://katahdin.mssm.edu/kismeth/primer_design.pl). 2×Taq Master Mix (Vazyme Biotech, Nanjing, China) was used for BS-PCR. The amplicon was cloned into pMD18-T (Takara Biotechnology, Dalian, China), and 10-14 positive clones per PCR product were sequenced by Biotechnology (Shanghai) Co., Ltd. The sequencing results were processed using the Seqman program of the DNASTAR software package to remove the vector and primer sequences and analyzed on the Kismeth website [31] . BnaIND.a-A3 was used to determine the conversion e ciency of bisul te [32] .
3. Results
3. Results
5
Identification of Endophytic Bacterial Strain
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Standard tests according to Bergey's Manual of Determinative Bacteriology 1994 were performed for the identi cation of the selected strain (WSS5). The genomic DNA of endophytic bacteria (WSS5) was extracted using the modi ed CTAB method (Doyle and Doyle, 1987) and the PCR ampli cation of 16S rDNA of the selected strain was done using bacterial universal primers, 27F
(5'AGAGTTTGATCMTGGCTCAG3') and 1492R (5'TACGGYTACCTTGTTACGACTT-3') (Lane 1991) . Ampli cation was done in an automated thermocycler (Eppendorf) with ampli cation steps as Preheating at 94°C for 3 min followed by 29 cycles with a denaturation step at 94°C for 30 sec, annealing step at 55°C for 1 min, and an extension step at 72°C for 1 min, followed by nal extension at72°C for 10 min.
The PCR product was puri ed using a Qiagen PCR Puri cation kit following manufacturer guidelines and was sequenced. To identify the closest neighbor of the selected isolate, their nucleotide sequences were subjected to Blast search in the NCBI GenBank database. A phylogenetic analysis was carried out by obtaining related sequences from the NCBI and an evolutionary tree was created using the neighborjoining analysis method and MEGA software, version 6.0, with 1000 bootstrap values (Tamura et al. 2013) .
(5'AGAGTTTGATCMTGGCTCAG3') and 1492R (5'TACGGYTACCTTGTTACGACTT-3') (Lane 1991) . Ampli cation was done in an automated thermocycler (Eppendorf) with ampli cation steps as Preheating at 94°C for 3 min followed by 29 cycles with a denaturation step at 94°C for 30 sec, annealing step at 55°C for 1 min, and an extension step at 72°C for 1 min, followed by nal extension at72°C for 10 min.
The PCR product was puri ed using a Qiagen PCR Puri cation kit following manufacturer guidelines and was sequenced. To identify the closest neighbor of the selected isolate, their nucleotide sequences were subjected to Blast search in the NCBI GenBank database. A phylogenetic analysis was carried out by obtaining related sequences from the NCBI and an evolutionary tree was created using the neighborjoining analysis method and MEGA software, version 6.0, with 1000 bootstrap values (Tamura et al. 2013) .
6
Identification of Endophytic Bacterial Strain
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Standard tests according to Bergey's Manual of Determinative Bacteriology 1994 were performed for the identi cation of the selected strain (WSS5). The genomic DNA of endophytic bacteria (WSS5) was extracted using the modi ed CTAB method (Doyle and Doyle, 1987) and the PCR ampli cation of 16S rDNA of the selected strain was done using bacterial universal primers, 27F
(5'AGAGTTTGATCMTGGCTCAG3') and 1492R (5'TACGGYTACCTTGTTACGACTT-3') (Lane 1991) . Ampli cation was done in an automated thermocycler (Eppendorf) with ampli cation steps as Preheating at 94°C for 3 min followed by 29 cycles with a denaturation step at 94°C for 30 sec, annealing step at 55°C for 1 min, and an extension step at 72°C for 1 min, followed by nal extension at72°C for 10 min.
The PCR product was puri ed using a Qiagen PCR Puri cation kit following manufacturer guidelines and was sequenced. To identify the closest neighbor of the selected isolate, their nucleotide sequences were subjected to Blast search in the NCBI GenBank database. A phylogenetic analysis was carried out by obtaining related sequences from the NCBI and an evolutionary tree was created using the neighborjoining analysis method and MEGA software, version 6.0, with 1000 bootstrap values (Tamura et al. 2013) .
(5'AGAGTTTGATCMTGGCTCAG3') and 1492R (5'TACGGYTACCTTGTTACGACTT-3') (Lane 1991) . Ampli cation was done in an automated thermocycler (Eppendorf) with ampli cation steps as Preheating at 94°C for 3 min followed by 29 cycles with a denaturation step at 94°C for 30 sec, annealing step at 55°C for 1 min, and an extension step at 72°C for 1 min, followed by nal extension at72°C for 10 min.
The PCR product was puri ed using a Qiagen PCR Puri cation kit following manufacturer guidelines and was sequenced. To identify the closest neighbor of the selected isolate, their nucleotide sequences were subjected to Blast search in the NCBI GenBank database. A phylogenetic analysis was carried out by obtaining related sequences from the NCBI and an evolutionary tree was created using the neighborjoining analysis method and MEGA software, version 6.0, with 1000 bootstrap values (Tamura et al. 2013) .
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