Dinh/Dinh 2013/NOTES/2013-1-3

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5hmC capture and sequencing project[edit]

  • As a proof of concept, we will use DMR330K probes to capture genomic DNA that have been processed for TAB-seq from Bing Ren's lab (in their 2012 publication, the authors published genome-wide hydroxymethylation sequencing of human ESCs/H1 and mouse ESCs).
  • Nearly all H1 5hmCs and mESCs 5hmCs exists in CG context (99.89% and 98.7%, respectively). These regions are significantly higher in distal-regulatory elements than in promoter proximal elements. TAB-seq found ~691K 5hmCs, whereas Tahilani et al (2009) estimated ~4.4million 5hmCs in human ESCs. Thus, there may be more 5hmC's which have not yet been characterized. Especially gene body 5hmCs (which were missing in TAB-seq, but found in affinity-based sequencing papers.)
  • Since 5hmC have strand bias, we need to use barcoded probes or random tagging so that we can remove clonal reads. We also need technical duplicates.
  • How many hmC sites (out of the total found genome-wide in hESCs) that can be characterized using DMR330K?
 Downloaded: GSM882245_H1.hmC_sites.FDR_0.0502.hg18.txt (from GEO), Yu et al dataset.
 Genome-wide sequencing characterized 691,414 5hmC sites in H1
 Our DMR330K covers 33,784 sites (~5%)
 Exp1-3:20713 (# sites covered by each set)
 Sub1  :  562
 Sub2  :  549
 Sub3  : 5225
 Sub4  : 5108
 Sub5  : 4033
  • We need to make sure that we get as much data as possible from the Exp1-3 probes set.
  • Next, we will determine how many additional potential hESCs 5hmC sites which may also be characterized with DMR330K (such as enhancer regions found in 10 tissues, they could be discovered during hESC differentiation or in cancer cells.)
  • Finally, we can measure probes performance and further optimize the probe design for a new probes set.
  • Since the gDNA given from Ren's lab is very precious, we need to establish the right testing conditions and controls before performing the actual experiments.

Plan:[edit]

 1) Learn how the experiment was performed in Yu et al 
 2) Develop testing conditions and controls for actual experiments 
 3) Obtain processed gDNA samples
 4) Perform bisulfite conversion, padlock capture, and sequencing

TAB-seq notes from Yu et al (2012)[edit]

  • Three key parameters determine the success of 5hmC detection:
 1) Efficient bisulfite conversion of unmodified cytosines to uracil (easy)
 2) Efficient bisulfite conversion of 5mC to 5caU/U (moderately easy)
 3) Efficient protection of 5hmC as 5gmC (most tricky?)
  • To access the conversion rates, the use of spiked-in lambda DNA amplified by PCR to contain 3 distinct domains with either unmodified cytosine, 5mC, or 5hmC. In their paper, Yu et al found that the most efficient steps were conversion of unmodified cytosines (>99%) to uracil and conversion of 5mC to 5caU/U (>97%). However, least efficient was hmC labeling to protect it from oxidation and bisulfite treatment (84-92%, maybe be higher due to 5% dCTP in dhmCTP when generating control DNA sequences).

Preparation of gDNA for TAB-seq (protocols taken directly from publication)[edit]

File:TAB-seq.jpg

  • (1) Glucosylation and Oxidation of Genomic DNA: Glucosylation reaction was performed in a 50 μl solution with 50 mM HEPES buffer (pH 8.0), 25 mM MgCl2, 100 ng/μl sonicated genomic DNA with spike-in control, 200 μM UDP-Glc, and 1 μM wild-type βGT. The reaction was incubated at 37°C for 1 hr. After the reaction, the DNA was purified by a QIAquick Nucleotide Removal Kit (QIAGEN). The oxidation reaction was performed in a 50 μl solution with 50 mM HEPES buffer (pH 8.0), 100 μM ammonium iron (II) sulfate, 1 mM α-ketoglutarate, 2 mM ascorbic acid, 2.5 mM DTT, 100 mM NaCl, 1.2 mM ATP, 10 ng/μl glucosylated DNA, and 3 μM recombinant mTet1. The reaction was incubated at 37°C for 1.5 hr. After proteinase K treatment, the DNA was purified with Micro Bio-Spin 30 Columns (Bio-Rad) and then by a QIAquick PCR Purification Kit (QIAGEN).
  • (2) Bisulfite conversion of treated gDNA or untreated gDNA: 500 ng–1 μg untreated or βGT/mTet1-treated (the same procedure as mouse ES/H1 cell) mouse cerebellum sample was applied to EpiTect Bisulfite Kit (QIAGEN) following the supplier's instruction.
  • (3) Library Generation: 500 ng–1 μg treated genomic DNA was end-repaired, adenylated, and ligated to methylated (5mC) adapters (Illumina TruSeq Genomic DNA adapters) according to standard Illumina protocols for genomic DNA library construction, maintaining the proper molar ratios of adaptor to insert. Adaptor ligated fragments with 200–600 bp inserts were gel purified by 2% agarose gel electrophoresis and sodium-bisulfite treated using the MethylCode kit (Invitrogen). Bisulfite-treated adaptor-ligated DNA was amplified by PCR with PfuTurbo Cx Hotstart DNA polymerase. The number of PCR cycles used was determined by quantification of bisulfite treated adaptor-ligated DNA by qPCR (KAPABiosystems library quant kit for Illumina libraries) such that the final library concentration obtained was approximately 20 nM. Final sequencing libraries were purified with AMPure XP beads or 2% agarose gel electrophoresis and quantified by qPCR (KAPABiosystems library quant kit for Illumina libraries). Up to three separate PCR reactions were performed per sample.

Preparation of spiked-in lambda DNA (protocols taken direct from publication)[edit]

  • IMPORTANT: We need to design & make probes to capture the spiked-in sequences: We need to make sure that the samples will have spiked-in controls (both control A for the methylC-Seq and control B for TAB-seq).
  • Generation of Spiked-in Conversion Controls for H1 Cells: Several spiked-in controls were generated and tested. Spike-in control A consisted of a 1:1 mixture of unmethylated lambda DNA (Promega Cat. No. D1521) with M.SssI-converted pUC19 DNA (NEB, Cat. No. M0226S). To generate the spiked-in control B, unmethylated lambda DNA (Promega Cat. No. D1521) was PCR amplified and purified by gel electrophoresis in non-overlapping 2 kb amplicons, with a cocktail of dATP/dGTP/dTTP and either: d5mCTP (Zymo Research, Cat. No. D1035) at genomic positions 0–10 kb, dCTP at genomic positions 20–30 kb, and d5hmCTP (Zymo Research, Cat. No. D1045) at genomic positions 38–48 kb. Amplicons with d5mCTP/d5hmCTP and dCTP were amplified by ZymoTaq DNA polymerase (Zymo Research, Cat. No. E2001) and Phusion HF DNA polymerase (NEB, Cat. No. M0530S), respectively, as per manufacturers' instructions. Spiked-in DNA was added to H1 genomic DNA to a final concentration of 0.5% (control A for replicate 1, control B for replicate 2), and sonicated to a range of 300–500 bp with a Biorupter 300 (high power, 15 s on, 15 s off, 20 cycles).

Preparation of catalytic mTet1[edit]

  • Ito et al 2010: Recombinant protein expression, purification and activity assays. For production of recombinant proteins, the catalytic domains of Tet1, Tet2 and Tet3 cDNAs were cloned into a modified pFastbac-HTb (Invitrogen) vector containing an N-terminal Flag tag. Generation of baculovirus that expresses Flag–Tet1(CD), Flag–Tet2(CD), or Flag–Tet3(CD) was performed using the Bac-to-Bac system (Invitorogen) according to the manufacturer’s instructions. The recombinant proteins were purified from infected Sf9 cells with the anti-Flag M2 antibody agarose affinity gel (Sigma-Aldrich) and eluted with buffer containing 10 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM DTT, 15% glycerol and 0.2 μg μl−1 Flag peptide.