The MCF10A progression series was purchased from the Barbara Ann Karmanos Cancer Institute and maintained in a culture of Dulbecco's modified Eagle's medium (DMEM) medium supplemented with 5% horse serum, 20 ng/mL EGF, 0.5 mg/mL hydrocortisone, 100 ng/mL cholera toxin, 10 µg/mL insulin and 1× antibiotic-antimycotic (Gibco, Grand Island, NY). All cell lines were verified by STR analysis and routinely screened for mycoplasma contamination.
2.6 Quantitative reverse transcription PCR primers
TaqMan assays were purchased from Thermo Fisher Scientific (Waltham, MA):, 18S (Hs99999901_s1), Actin (Hs99999903_m1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Hs02758991_g1), HOTAIR (Hs03296680). Sybr‐green assays purchased from Integrated DNA Technologies (Coralville, IA):
AC053503.6 Forward Primer: 5’-AGGTGGATTAGAGGGGGTGT-3’
Reverse Primer: 5’-GGCTGAGAAGGGGGTTTCTG-3’,
AC068580.7 Forward Primer: 5’-CCCGTCGTGACCTCATTGTG-3'
Reverse Primer: 5’-GAACCCCTTTTCCTCACCCA-3’,
CCND2-AS1 Forward Primer: 5’-CAAGCTGGAACCCTGCAAGA-3’
Reverse Primer: 5’-AAGGGTATACCTTCCTCCCCA-3’,
CTD-2033D15.1 Forward Primer: 5’-GGTAAGAAGCAAAGCCCTGGA3’
Reverse Primer: 5’-TGGCTGAGACGCCATCTGTA-3’,
FAM83H-AS1 Forward Primer: 5’-GCAACACCCTACTGACCTTGT-3’
Reverse Primer: 5’-AGCTCTGTGGTGACTGTCTT-3’,
FAM87A Forward Primer: 5’-TTCCGCAGGTTTTAGTGGCT-3’
Reverse Primer: 5’-CAAACTGTCCCCAACTCCCA-3’,
GATA2-AS1 Forward Primer: 5’-GACCCTCTGAAAGACACCGC-3’
Reverse Primer: 5’-TCTTGCTCATGTGTGAGGGC-3’,
HCG9 Forward Primer: 5’-CAGGAACCCAGGGACTTCAG-3’
Reverse Primer: 5’-TGTTCTCTGCAGCTTGACCT-3’,
HOXA11-AS Forward Primer: 5’-TCCGATTTGCACGGTGACTT-3’
Reverse Primer: 5’-CGGATGTCAGCGCCTCTAAA-3’,
LINC00885 Forward Primer: 5’-GGCACTGTAGAAGCCCCATT-3’
Reverse Primer: 5’-GTCCAGCGAACTGAAGGACA-3’,
LINC01125 Forward Primer: 5’-AGGCAAAGATGAGCAGAGCC-3’
Reverse Primer: 5’-CCAAGCAATGCTGGTTCCTTT-3’,
LINC01589 Forward Primer: 5’-AAATGGAATGCAGCCACACC-3’
Reverse Primer: 5’-CCAAGAGGCCATCCGTCTTC-3’,
NR2F1-AS1 Forward Primer: 5’-GGTCACGGAGAAAACAGGTTCA-3’
Reverse Primer: 5’-CCCCAGAGCTGCATCCTTATG-3’
OSMR-AS1 Forward Primer: 5’-TTGGAAACCGAAAACTCGGC-3’
Reverse Primer: 5’-ACATTGGGATGTTCTGCCCC-3’,
PCAT1 Forward Primer: 5’-CCTCTAAGTGCCAGTGCAGG-3’
Reverse Primer: 5’-ATGTATCTGCGCACCCTTTGA-3’,
RP11-107N15.1 Forward Primer: 5’-GGGTCCTCAATGTGGGGTTT-3’
Reverse Primer: 5’-TCGCTAGAGTCACCCCAGTT-3’,
RP11-258F1.1 Forward Primer: 5’-CGTTGTACAGGCCCTTCTCA-3’
Reverse Primer: 5’-GTGCGCACAACCCTGGTATC-3’,
RP11-303E16.3 Forward Primer: 5’-CAGACTCCGTACGCCTTCAC-3’
Reverse Primer: 5’-CTGAGCCTGCAACTCGACTG-3’,
RP11-323N12.5 Forward Primer: 5’-TGGACCAGTCGAAACCCTTG-3’
Reverse Primer: 5’-TCTCGACATCGAGGACCCAT-3’,
RP11-326G21.1 Forward Primer: 5’-ACTCCGCATTACACCACTGA-3’
Reverse Primer: 5’-CCCGAAACAGTACCAGGCAA-3’,
RP11-346D6.6 Forward Primer: 5’-CAAGCAGCCCTGGAGAGTTTA-3’
Reverse Primer: 5’-AACTTGGGGGTCACAGCATC-3’
RP11-373D23.3 Forward Primer: 5’-CTTCCAAGGCCCTGCATGAT-3’
Reverse Primer: 5’-GGTGAGGGAAGACAACACGG-3’,
RP11-379F4.4 Forward Primer: 5’-TGCCCGGTTTTATAGCCCTG-3’
Reverse Primer: 5’-ATCTGTTCCGTGCTCCCTTC-3’,
RP11-403A21.1 Forward Primer: 5’-AGGGATGGGGTCTCGAGTTT-3’
Reverse Primer: 5’-TCAGCTGGTGGGTGTTTAGC-3’,
RP11-465B22.8 Forward Primer: 5’-AGCCTGAGCTCATCCAACAC-3’
Reverse Primer: 5’-GTGCGTGAACTGCAGACTTT-3’
RP3-483K16.4 Forward Primer: 5-’AGTTGCCATTGAGCTCCACAA-3’
Reverse Primer: 5’-TGGACTACTGGCAGAAGCGT-3’,
RP11-507M3.1 Forward Primer: 5’-CGCATTTTCCTGATTGGCCC-3’
Reverse Primer: 5’-ACATTCCCCTTCAACGCCTG-3’,
RP11-560J1.2 Forward Primer: 5’-CCTAGGGTAGTCCGAGGTCA-3’
Reverse Primer: 5’ACAAAATACGCCCGGCAAAG-3’,
RP11-61F12.1 Forward Primer: 5’-GGACGTGGTTTGCTAGGTGA-3’
Reverse Primer: 5’-ACAGGTTTTCCGTCTCCGAC-3’,
RP11-63G10.2 Forward Primer: 5’-ACCTGTGCCAGTGTGAACAA-3’
Reverse Primer: 5’-GGGCTAGTCAAAGTCAGCGT-3’,
SLC44A3-AS1 Forward Primer: 5’-AGCAACAGTGTAGTGGCGTA-3’
Reverse Primer: 5’-CTGGCCTGTGATGCTTTTCC-3’,
SNHG18 Forward Primer: 5’-CATGTTCCCAGAGGTTGGCA-3’
Reverse Primer: 5’-AGAGGACAAGGCAAAACACTT-3’,
TMEM220-AS1 Forward Primer: 5’-TCCAAGTCCCCTTCTGACTTC-3’
Reverse Primer: 5’-CAGGCTCCTCAGGAAGAATCC-3’
SNORD3B-2 Forward Primer: 5’-GGCAGTGTAGCGAGAAAGGT-3’
Reverse Primer: 5’-AATAGGAGGTGCCACACAGC-3’
2.7 Chromatin Immunoprecipitation (ChIP)
MCF10A, MCF10A-AT1, DCIS.com, and MCF10A-CA1 cells were grown to a final count of 5 x 10^6. Cells were chemically crosslinked by the addition of 1 mL of fresh 10% formaldehyde solution for 10 min at room temperature on a rocker. After 10 minutes added 1/10 volume of 1.25M Glycine to quench unreacted formaldehyde and incubated for 5 minutes on rocker. Cells were pelleted at 1000g for 5 minutes, washed twice with 1 × PBS, flash frozen in liquid nitrogen, and stored at − 80°C prior to use. Cells were resuspended, lysed in lysis buffer (50 mM HEPES, 140 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% Sodium deoxycholate, .1% SDS), and sonicated to solubilize and shear crosslinked DNA. Sonication conditions vary depending on cells, but cells were sonicated using a Diagenode Bioruptor Sonicator and sonicated at power 7 for 13 × 30s pulses (30s pause between pulses) at 4°C while samples were immersed in an ice bath. The sonicated cells were centrifuged for 10 min at 8000g at 4°C and the supernatant collected to proceed with immunoprecipitation. The resulting whole-cell extract volume was divided into two, one for IgG and the other for H3K27ac targeting. The samples were diluted in 1:10 ratio with RIPA buffer (50 mM Tris-HCl pH8, 150 mM NaCl, 2 mM EDTA pH8, 1% NP-40, 0.5% Sodium Deoxycholate, 0.1% SDS) incubated overnight at 4°C with the 2.5 µg of the appropriate antibody, Cell Signaling IgG (Rabbit (DA1E) mAb IgG XP® Isotype Control #3900) and Abcam H3K27ac (Anti-Histone H3 (acetyl K27) antibody - ChIP Grade (ab4729)). The following day, 60 µL of ChIP Grade Protein G Magnetic beads (Cell Signaling 9006S) were washed three times with RIPA buffer and 30 µL each of the washed beads were added to IgG and H3K27ac samples and left rotating at 4°C for 3 hours. IgG and H3K27ac samples with magnetic beads were then washed three times with low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl) and one time with high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl). DNA was then eluted off the beads for each sample by heating at 65°C at 1200g for 1 hour, cooling each sample at room temp for 2 min, centrifuging for 1 min at 10,000g, and putting each sample on a magnet for 2 min and removing the liquid. 4.8 µL of 5 M NaCl and 2 µL RNase A (10 mg/mL) was added to each sample and incubated while shaking at 1200rpm, 65°C overnight. The next day, 2 µL proteinase K (20 mg/mL) was added to each sample and incubated while shaking at 1400 rpm, 60°C for 1 hour. DNA was purified using a QIAGEN QIAquick PCR purification kit (cat. Number 28104).