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Резюме

We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.

Аннотация

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical disease or phenotype. For many diseases and traits, most variants are non-coding, and are thus likely to act by impacting subtle, comparatively hard to predict mechanisms controlling gene expression. Here, we describe a general strategic approach to prioritize non-coding variants, and screen them for their function. This approach involves computational prioritization using functional genomic databases followed by experimental analysis of differential binding of transcription factors (TFs) to risk and non-risk alleles. For both electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genetic variants, a synthetic DNA oligonucleotide (oligo) is used to identify factors in the nuclear lysate of disease or phenotype-relevant cells. For EMSA, the oligonucleotides with or without bound nuclear factors (often TFs) are analyzed by non-denaturing electrophoresis on a tris-borate-EDTA (TBE) polyacrylamide gel. For DAPA, the oligonucleotides are bound to a magnetic column and the nuclear factors that specifically bind the DNA sequence are eluted and analyzed through mass spectrometry or with a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. This general approach can be widely used to study the function of non-coding genetic variants associated with any disease, trait, or phenotype.

Введение

Секвенирования и генотипирования на основе исследований, включая геномные исследования ассоциации (GWAS), кандидат исследований локуса, и глубокие секвенирования исследования, выявили множество генетических вариантов, которые статистически связаны с заболеванием, признаком, или фенотипа. Вопреки ранних предсказаний, большинство из этих вариантов (85-93%) расположены в некодирующих областей и не изменяют аминокислотную последовательность белков 1,2. Интерпретируя функции этих некодирующих вариантов и определения биологических механизмов , связывающих их с сопутствующей болезни, признак или фенотип оказался непростым 3-6. Мы разработали общую стратегию для выявления молекулярных механизмов, связывающих варианты к важному промежуточного фенотипа - экспрессии генов. Этот трубопровод разработан специально для идентификации модуляции TF связывания генетических вариантов. Эта стратегия сочетает в себе вычислительные подходы и методы молекулярной биологии, направленных предсказатьбиологические эффекты вариантов кандидатов в силикомарганца, и проверить эти предсказания эмпирически (рисунок 1).

figure-introduction-1204
Рисунок 1:.. Стратегический подход для анализа некодирующих генетические варианты шагов, которые не включены в подробный протокол , связанный с этой рукописи заштрихованы серым цветом Пожалуйста , нажмите здесь , чтобы посмотреть увеличенную версию этой фигуры.

Во многих случаях, важно, чтобы начать путем расширения списка вариантов, чтобы включить все те, в высокой рычажной-неравновесия (LD) с каждым статистически связанным вариантом. ЛД является мерой неслучайной ассоциации аллелей двух различных хромосомных положениях, которые могут быть измерены с помощью R 2 статистики 7. R 2 представляет собой меру линКейдж неравновесие между двумя вариантами, с R 2 = 1 , обозначающее совершенной связи между двумя вариантами. Аллели в высокой LD оказываются совместно разделять на хромосоме через родовые популяции. Современные генотипирования массивы не включают в себя все известные варианты в геноме человека. Вместо этого они используют ЛД внутри генома человека и включают в себя подмножество известных вариантов , которые действуют в качестве прокси для других вариантов в пределах определенной области LD 8. Таким образом, вариант без какого-либо биологического последствие может быть связан с конкретным заболеванием, потому что это в LD с причинным вариантом: вариант с значимого биологического эффекта. С процедурной точки зрения рекомендуется преобразовать последний выпуск 1000 геномов проекта 9 файлов вариант вызова (VCF) в двоичные файлы , совместимые с Plink 10,11, инструмент с открытым исходным кодом для анализа всего генома ассоциации. Впоследствии все другие генетические варианты с LD г 2> 0,8 с каждым входом генетической ваRiant могут быть идентифицированы в качестве кандидатов. Важно использовать соответствующий ссылочный населения для этого , например , Step - , если вариант был выявлен у пациентов европейского происхождения, данные от субъектов аналогичного происхождения должны быть использованы для расширения LD.

Расширение LD часто приводит десятки вариантов кандидатов, и вполне вероятно, что лишь небольшая часть из них внести свой вклад в механизм заболевания. Часто, это неосуществимо экспериментально исследовать каждый из этих вариантов по отдельности. Поэтому полезно использовать тысячи доступных функциональных публично геномных наборов данных в качестве фильтра для определения приоритетности вариантов. Например, КОДИРОВАНИЯ консорциум 12 выполнил тысячи чиповых-сл экспериментов , описывающих связывание ТФ и сопутствующих факторов и гистонов марок в широком диапазоне контекстов, наряду с данными хроматина доступности от технологий , таких как ДНКазы сл 13, ATAC -seq 14 и FAIRE-15 сл. Databтузы и веб - серверы , такие как браузера УСК генома 16, Дорожная карта Epigenomics 17 Blueprint эпигеном 18, Cistrome 19 и ReMap 20 обеспечивают свободный доступ к данным , полученных с помощью этих и других экспериментальных методов в широком диапазоне типов и условий клеток. Когда есть слишком много вариантов, чтобы проверить экспериментально, эти данные могут быть использованы для приоритеты тех, которые находятся в пределах возможных регуляторных областей в соответствующих тканей и клеток типов. Кроме того, в тех случаях, когда вариант в микросхеме-сл пика для специфического белка, эти данные могут обеспечить потенциальных клиентов, как к специфическому TF (ами) или кофакторов, связывание может затрагивающий.

Далее, полученные по приоритетам варианты экспериментально скринингу для проверки связывания с использованием EMSA 21,22 предсказанную генотип-зависимого белка. EMSA измеряет изменение миграции олиго на невосстановительной геле КЭ. Флуоресцентно меченных олиго инкубируют сядерный лизат, и связывание ядерных факторов замедлит движение олиго на геле. Таким образом, олиго-, который связан больше ядерных факторов представит в качестве более сильного флуоресцентного сигнала при сканировании. Следует отметить, что EMSA не требует предсказания о специфических белков, связывание будут затронуты.

После того, как варианты идентифицируются, которые расположены в пределах прогнозируемых регуляторных областей и способны дифференцированно связывания ядерных факторов, вычислительные методы используются для прогнозирования конкретный TF (ы), связывание которого они могут повлиять. Мы предпочитаем использовать CIS-BP 23,24, RegulomeDB 25, UNIProbe 26 и 27 Джаспер. После того, как кандидат ТФ определены, эти прогнозы могут быть специально протестированы с использованием антител против этих ТФ (EMSA-supershifts и Dapa-вестерны). EMSA-supershift включает добавление антитела к TF специфичные к ядерной лизата и олиго. Положительный результат в EMSA-supershift является магнезииesented как дальнейший сдвиг в полосе EMSA или потери полосы (обзор в ссылке 28). В дополнительном Dapa, 5'-биотинилированные олиго дуплекс, содержащий вариант и 20 пар оснований нуклеотидов фланговый инкубируют с ядерными лизата из соответствующего типа (ов) клеток, чтобы захватить любые ядерные факторы, специфически связывающих олигонуклеотидов. Фактор-комплекс олиго дуплексной ядерная скованной стрептавидином микрогранулы в магнитном колонке. Связанные ядерные факторы собираются непосредственно через элюирования 29,48. Связующие предсказания затем могут быть оценены с помощью Вестерн-блоттинга с использованием антител, специфичных к белку. В тех случаях, когда нет никаких очевидных предсказаний, или слишком много прогнозов, то элюции из вариантов отжимания экспериментов Dapa могут быть отправлены в активную зону протеомики для выявления кандидатов ТФ с использованием масс-спектрометрии, которые впоследствии могут быть проверены с помощью этих ранее описанных методы.

В оставшейся части статьи обе, подробный протокол для EMSA и Dapa анализа генетических вариантов предусмотрена.

протокол

1. Preparation of Solutions and Reagents

  1. Order custom DNA oligonucleotide probes for use in EMSA and DAPA.
    1. To reduce non-specific protein binding, design short oligos (between 35-45 base pairs (bp) in length)30, and place the variant of interest directly in the center flanked by its 17 bp endogenous genomic sequence. For EMSA oligos, add a 5' fluorophore. For DAPA oligos, add a 5' biotin tag.
    2. Order both the sense strand and its reverse complement strand. Alternatively, order duplex (pre-annealed) oligos. When naming the oligos, base the nomenclature on an established reference genome.
      Note: "Risk" and "non-risk" designation can be disease and project specific, while "reference" and "non-reference" are more universally relevant.
    3. Upon arrival of the oligos, briefly spin down the contents and resuspend in nuclease-free water to a final concentration of 100 µM. Store resuspended stock at -20 °C. Protect oligos tagged with a fluorophore from light by wrapping with aluminum foil.
NameSequence
rs76562819_REF_FORGTAATGCCTTAATGAGAGAGAGTTAGTCATCTTCTCACTTC
rs76562819_REF_REVGAAGTGAGAAGATGACTAACTCTCTCTCATTAAGGCATTAC
rs76562819_NONREF_FORGTAATGCCTTAATGAGAGAGGGTTAGTCATCTTCTCACTTC
rs76562819_NONREF_REVGAAGTGAGAAGATGACTAACCCTCTCTCATTAAGGCATTAC

Table 1: Example EMSA/DAPA oligonucleotide design to test a SNP for differential binding. "REF" stands for the reference allele, while "NONREF" stands for the non-reference allele. "FOR" stands for the forward strand, while "REV" indicates its complement. The SNP is seen in red.

  1. Prepare cytoplasmic extraction (CE) buffer with a final concentration of 10 mM HEPES (pH 7.9), 10 mM KCl, and 0.1 mM EDTA in deionized water.
  2. Prepare nuclear extraction (NE) buffer with a final concentration of 20 mM HEPES (pH 7.9), 0.4 M NaCl, and 1 mM EDTA in deionized water.
  3. Prepare annealing buffer with a final concentration of 10 mM Tris (pH 7.5-8.0), 50 mM NaCl, and 1 mM EDTA in deionized water.

2. Preparation of Nuclear Lysate from Cultured Cells

Note: This experimental protocol was optimized using B-lymphoblastoid cell lines, but has been tested in several other unrelated adherent/ suspension cell lines and works universally.

  1. Culture B-lymphoblastoid cells in Roswell Park Memorial Institute (RPMI) 1640 with 2 mM L-glutamine, 10% fetal bovine serum, and 1x antibiotic-antimycotic containing 100 units/ml of penicillin, 100 µg/ml of streptomycin, and 250 ng/ml of amphotericin B.
    1. Seed at a range of 200,000-500,000 viable cells/ml and incubate flasks at 37 °C with 5% carbon dioxide in an upright position with vented or loose caps.
      Note: The growth of B-lymphoblastoid cells slows when they reach over 1,000,000 cells/ml. Break up cell blasts by pipetting up and down several times and return cells to 200,000-500,000 cells/ml to maintain a rapid rate of growth.
  2. Wash cultured cells twice with 10 ml ice cold phosphate buffered saline (PBS), spin down at 4 °C, 300 x g for 5 min and remove PBS via aspiration.
  3. Count cells using a hemocytometer and resuspend pellet as 1 ml ice cold PBS per 107 cells.
    Note: For example, if lysing 2 x 107 cells, resuspend in 2 ml PBS.
  4. Aliquot 1 ml to 1.5 ml microcentrifuge tubes so that each tube contains 107 cells in PBS. Centrifuge at 3,300 x g for 2 min 4 °C and aspirate off PBS.
  5. Prior to use, add 1 mM dithiothreitol (DTT), 1x phosphatase inhibitor, and 1x protease inhibitor to a working stock of CE buffer. Resuspend cell pellet with 400 µl of CE buffer and incubate on ice for 15 minutes.
  6. Add 25 µl of 10% Nonidet P-40 and mix by pipetting. Centrifuge at 4 °C, max speed for 3 min. Decant and discard the supernatant.
  7. Prior to use, add 1 mM DTT, 1x phosphatase inhibitor, and 1x protease inhibitor to a working stock of NE buffer. Resuspend cell pellet with 30 µl of NE buffer and mix by vortexing.
  8. Incubate at 4 °C in a tube rotator or on ice for 10 min. Centrifuge 3,300 x g for 2 min 4 °C.
  9. Collect the clear supernatant (nuclear lysate) and aliquot before storing at -80 °C to avoid multiple freeze-thaw cycles that may degrade the protein. Leave a 10 µl aliquot to measure protein concentration using the bichoninic acid assay (BCA)31.

3. Electrophoretic Mobility Shift Assay (EMSA)

  1. Prepare Oligo Working Stock and EMSA Gel.
    1. If oligos were ordered in duplex, thaw the 100 µM stock and dilute 1:2,000 in annealing buffer to achieve a 50 nM working stock.
    2. If oligos were ordered single-stranded, thaw the 100 µM stocks and dilute 1:10 in annealing buffer to achieve 100 nM working stocks. Combine 100 µl of the 100 nM complement strand solution with each other in a microcentrifuge tube.
      1. Place in a heat block at 95 °C for 5 min. Turn off the heat block and allow the oligos to slowly cool down to room temperature for at least one hour prior to use.
    3. Pre-run the EMSA Gel.
      1. Remove the slide from a pre-cast 6% TBE gel and rinse under deionized water several times to remove any buffer from the wells. Prepare 1 L of 0.5x TBE buffer by adding 50 mL of 10x TBE to 950 ml of deionized water.
      2. Assemble the gel electrophoresis apparatus and check for leaks by filling the inner chamber with 0.5x TBE buffer. If no buffer leaks into the outer chamber, fill the outer chamber roughly two thirds of the way.
      3. Pre-run the gel at 100 V for 60 min.
      4. Flush each well with 200 µl of 0.5x TBE buffer.
  2. Prepare Binding Buffer Master Mix.
    1. Prepare 10x binding buffer with a final concentration of 100 mM Tris, 500 mM KCl, 10 mM DTT; pH 7.5 in deionized water.
    2. In a microcentrifuge tube, create a master mix consisting of the reagents common to all reactions (10 µl 10x binding buffer, 10 µl DTT/polysorbate, 5 µl Poly d(I-C), and 2.5 µl salmon sperm DNA; Table 2). Prepare an additional 10% to account for volume loss due to pipetting.
ReagentFinal Conc.Rxn #1Rxn #2Rxn #3Rxn #4
Ultrapure Waterto 20 µl vol.13.5 µl11.98 µl13.5µl11.98 µl
10x Binding Buffer1x2 μl2 μl2 μl2 µl
DTT/TW-201x2 μl2 μl2 μl2 μl
Salmon Sperm DNA500 ng/μl0.5 μl0.5 μl0.5 μl0.5 μl
1μg/μl Poly d(I-C)1 μg1 μl1 μl1 μl1 μl
Nuclear Extract (5.26 ug/µl)8 μg-1.52 μl-1.52 μl
NE Buffer1.52 μl-1.52 μl-
Reference allele oligo50 fmol1 μl1 μl--
Non-Reference allele oligo50 fmol--1 μl1 μl

Table 2: Example EMSA reaction setup. The table illustrates an example EMSA to test the hypothesis that there is genotype-dependent binding of TFs to a specific SNP.

  1. Add nuclease-free water to each microcentrifuge tube such that the final volume following addition of all reagents will be 20 µl.
  2. Add the appropriate amount (5.5 µl) of master mix to each microcentrifuge tube.
  3. Add 8 µg of nuclear lysate to the appropriate microcentrifuge tubes. Include tubes containing the oligo without nuclear extract as negative controls (e.g. Table 2, Rxn #1 and Rxn #3).
    Note: The optimal amount of lysate per reaction must be determined experimentally by titration. Generally, titrating a range of 2-10 µg of lysate is sufficient.
  4. Add 50 fmol of oligo to the appropriate microcentrifuge tubes. Flick to mix and briefly spin the contents to the bottom of the tube. Incubate for 20 min at room temperature.
    Note: If attempting a supershift, incubate the lysate mixture with antibody for 20 min at room temperature prior to the addition of oligos. It is recommended to use 1 µg of a ChIP-grade antibody for best results.
  5. Add 2 µl of 10x Orange Loading Dye to each microcentrifuge tube. Pipette up and down to mix.
  6. Load the samples into the pre-run 6% TBE gel by pipetting up and down to mix and then expelling each sample into a separate well. Run the gel at 80 V until the orange dye has migrated 2/3 to 3/4 of the way down the gel. This should take approximately 60-75 min.
  7. Remove the gel from the plastic cassette by prying it open with a gel knife and place the gel in a container with 0.5% TBE buffer to keep it from drying out.
  8. Place the gel on the surface of an infrared and chemiluminescence imaging system, being sure to eliminate any bubbles or contaminants that will disrupt the image.
  9. Using the scanning system software, click the "Acquire" tab and then select "Draw New" to draw a box around the area corresponding to where the gel is located on the surface of the scanner.
  10. In the "Channels" section of the "Acquire" tab, select the channel corresponding to the wavelength of the fluorophore tag on the oligo. In the "Scanner" section, click "preview" to get a low-quality preview scan. Adjust the scan area by dragging the blue box surrounding the acquired preview image down to the portion of the gel to be imaged.
    Note: For example, if using oligos labeled with a 700 nm fluorophore, make sure the "700 nm" channel is selected before scanning.
  11. In the "Scan Controls" section, select the "84 µM" resolution option and the "Medium" quality option. Set the focus to offset to half the thickness of the gel. Note: For example, a 1 mm gel would use a 0.5 mm focus offset.
  12. In the "Scanner" section, click "Start" to begin the scan.
    Note: During the scan, the brightness, contrast, and color scheme can often be adjusted manually depending on the manufacturer of the scanning system.
  13. After the scan has finished, select the "Image" tab and click "Rotate or Flip" in the "Create" section to correct the orientation. Save the image file by clicking "Export" in the main menu and then select "Single Image View."

4. DNA Affinity Purification Assay (DAPA)

  1. Preparation of 5 µM Oligo Working Stock.
    1. If oligos were ordered in duplex, thaw the 100 µM stock and dilute 1:20 in annealing buffer to achieve a 5 µM working stock.
    2. If oligos were ordered single-stranded, thaw the 100 µM stocks and dilute 1:10 in annealing buffer to achieve 10 µM working stocks. Combine 10 µl of the 10 µM complementary strands with each other. Place in a heat block at 95 °C for 5 min. Turn off the heat block and allow the oligos to slowly cool down to room temperature prior to use.
  2. Before starting, warm the binding buffer, low stringency wash buffer, high stringency wash buffer, and elution buffer to room temperature.
    Note: A final concentration of 50 ng/mL Poly d(I-C) can be added to the binding buffer, low stringency wash buffer, and high stringency wash buffer to reduce potential non-specific binding of proteins to the oligos.
  3. Prepare the binding mixtures for each variant.
    1. Mix 1 volume of nuclear lysate with 2 volumes of binding buffer.
      Note: The required amount of lysate must be determined experimentally due to varying abundance of TFs. Using between 100-250 µg of nuclear lysate per column is sufficient in most cases.
    2. Add 1x phosphatase inhibitor, 1x protease inhibitor, and 1x binding enhancer (optional) and mix by flicking the tube several times.
      Note: 100x binding enhancer consists of 750 mM MgCl2 and 300mM ZnCl2. Add binding enhancer if the binding of the TF to DNA depends on cofactors or reducing agents. If this information is not known, add the binding enhancer.
    3. Add 10 µl of 5 µM biotinylated capture DNA (50 pmol) to each respective binding mixture. Incubate for 20 min at room temperature.
      Note: Incubation time and temperature may vary depending on the TF. The optimal values need to be determined experimentally.
  4. Add 100 µl of streptavidin microbeads. Incubate for 10 min at room temperature.
  5. For each oligo probe being tested, place a binding column in the magnetic separator. Place a microcentrifuge tube directly under each binding column and apply 100 µl of binding buffer to rinse the column.
  6. Pipette the contents of each binding mixture into separate columns and allow the liquid to flow completely through the column into the microcentrifuge tube before proceeding. Make sure to label the columns with the variant oligo that was used in the binding mixture. Label the flow-through samples and replace with new microcentrifuge tubes to collect the low-stringency washes.
  7. Apply 100 µl of low-stringency wash buffer to the column; wait until the column reservoir is empty. Repeat wash 4x. Label the low-stringency wash samples and replace with new microcentrifuge tubes to collect the high-stringency washes.
  8. Apply 100 µl of high-stringency wash buffer to the column; wait until the column reservoir is empty. Repeat wash 4x. Label the high-stringency wash samples and replace with new microcentrifuge tubes to collect the pre-elution.
  9. Add 30 µl of native elution buffer to the column and let stand for 5 min. Label the pre-elution samples and replace with new microcentrifuge tubes to collect the elution.
    Note: This does not elute the bound protein; it washes the remaining high-stringency buffer out of the column and replaces it with elution buffer to maximize the efficiency of the elution.
  10. Add an additional 50 µl native elution buffer to elute the bound TFs. For a higher yield but less concentrated eluate, add an additional 50 µl of native elution buffer and collect the flow-through.
    Note: Analyze elution samples through mass spectrometry to determine the identity of the bound TFs32. Afterwards, verify the proteomic results through sodium dodecyl sulfite polyacrylamide gel electrophoresis (SDS-PAGE) followed by a Western blot33. If mass spectrometry is not available, run a silver stain using standard technique instead of a Western blot to determine the size of the protein(s) showing genotype-dependent binding. Use this information to narrow down the list of predicted TFs from the computational approaches detailed in the introduction.

Результаты

In this section, representative results of what to expect are provided when performing an EMSA or DAPA, and the variability with regards to the quality of lysate is characterized. For example, it has been suggested that freezing and thawing protein samples multiple times may result in denaturation. In order to explore the reproducibility of EMSA analysis in the context of these "freeze-thaw" cycles, two 35 bp oligos differing at one genetic variant were incubated with a single bat...

Обсуждение

Although advances in sequencing and genotyping technologies have greatly enhanced our capacity to identify genetic variants associated with disease, our ability to understand the functional mechanisms impacted by these variants is lagging. A major source of the problem is that many disease-associated variants are located in n on-coding regions of the genome, which likely affect harder-to-predict mechanisms controlling gene expression. Here, we present a protocol based on the EMSA and DAPA techniques, valuable molecular t...

Раскрытие информации

The authors have nothing to disclose.

Благодарности

We thank Erin Zoller, Jessica Bene, and Lindsey Hays for input and direction in protocol development. MTW was supported in part by NIH R21 HG008186 and a Trustee Award grant from the Cincinnati Children's Hospital Research Foundation. ZHP was supported in part by T32 GM063483-13.

Материалы

NameCompanyCatalog NumberComments
Custom DNA OligonucleotidesIntegrated DNA Technologieshttp://www.idtdna.com/site/order/oligoentry
Potassium ChlorideFisher ScientificBP366-500KCl, for CE buffer
HEPES (1 M)Fisher Scientific15630-080For CE and NE buffer
EDTA (0.5M), pH 8.0Life TechnologiesR1021For CE, NE, and annealing buffer
Sodium ChlorideFisher ScientificBP358-1NaCl, for NE buffer
Tris-HCl (1M), pH 8.0InvitrogenBP1756-100For annealing buffer
Phosphate Buffered Saline (1x)Fisher ScientificMT21040CMPBS, for cell wash
DL-Dithiothreitol solution (1 M)Sigma646563Reducing agent
Protease Inhibitor CocktailThermo Scientific87786Prevents catabolism of TFs
Phosphatase Inhibitor Cocktail Thermo Scientific78420Prevents dephosphorylation of TFs
Nonidet P-40 SubstituteIBI ScientificIB01140NP-40, for nuclear extraction
BCA Protein Assay KitThermo Scientific23225For measuring protein concentration
Odyssey EMSA Buffer KitLicor829-07910Contains all necessary EMSA buffers
TBE Gels, 6%, 12 WellsInvitrogenEC6265BOXFor EMSA
TBE Buffer (10x)Thermo ScientificB52For EMSA
FactorFinder Starting KitMiltenyi Biotec130-092-318Contains all necessary DAPA buffers
Licor Odyssey CLxLicorRecommended scanner for DAPA/EMSA
Antibiotic-AntimycoticGibco15240-062Contains 10,000 units/ml of penicillin, 10,000 µg/ml of streptomycin, and 25 µg/ml of Fungizone® Antimycotic
Fetal Bovine SerumGibco26140-079FBS, for culture media
RPMI 1640 MediumGibco22400-071Contains L-glutamine and 25 mM HEPES

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Functional Non coding Genetic VariantsElectrophoretic Mobility Shift Assay EMSADNA affinity Precipitation Assay DAPARegulatory ProteinsPathwaysB lymphoblastoid CellsNuclear LysatesCE BufferNE bufferDithiothreitol DTTPhosphatase InhibitorPhenylmethanesulfonyl Fluoride PMSFNonidet P 40

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