cell type: Lin (CD2, CD3, CD4, CD7, CD10, CD11b, CD14, CD15, CD19, CD20, CD56, and Glycophorin A )-, CD34+, CD38-, CD90+ cells disease status: healthy restriction enzyme: MspI
Treatment protocol
No treatment.
Growth protocol
Uncultured primary cells.
Extracted molecule
genomic DNA
Extraction protocol
Cells were sorted into RLT buffer (Qiagen, Valencia, CA), homogenized by passing five times through a needle. Simultaneous harvest of RNA and genomic DNA was achieved with the AllPrep kit (Qiagen, Valencia, CA) following the instructions of the manufacturer. Integrity of genomic DNA of high molecular weight was assured by electrophoresis for all samples used. HpaII or MspI (NEB, Ipswich, MA) digestions of genomic DNA were performed overnight prior to overnight ligation of the HpaII adapter with T4 ligase. PCR amplified adapter-ligated HpaII or MspI fragments were submitted to Roche-NimbleGen, Inc. (Madison, WI) and labeled according to protocol (see manufacturer's web site www.nimblegen.com).
Label
Cy3
Label protocol
Labeling was carried out by Roche/Nimblegen (Madison, WI). For details, see manufacturer's web site www.nimblegen.com.
Channel 2
Source name
sorted LT-HSC from human bone marrow, HpaII-digested DNA
cell type: Lin (CD2, CD3, CD4, CD7, CD10, CD11b, CD14, CD15, CD19, CD20, CD56, and Glycophorin A )-, CD34+, CD38-, CD90+ cells disease status: healthy restriction enzyme: HpaII
Treatment protocol
No treatment.
Growth protocol
Uncultured primary cells.
Extracted molecule
genomic DNA
Extraction protocol
Cells were sorted into RLT buffer (Qiagen, Valencia, CA), homogenized by passing five times through a needle. Simultaneous harvest of RNA and genomic DNA was achieved with the AllPrep kit (Qiagen, Valencia, CA) following the instructions of the manufacturer. Integrity of genomic DNA of high molecular weight was assured by electrophoresis for all samples used. HpaII or MspI (NEB, Ipswich, MA) digestions of genomic DNA were performed overnight prior to overnight ligation of the HpaII adapter with T4 ligase. PCR amplified adapter-ligated HpaII or MspI fragments were submitted to Roche-NimbleGen, Inc. (Madison, WI) and labeled according to protocol (see manufacturer's web site www.nimblegen.com).
Label
Cy5
Label protocol
Labeling was carried out by Roche/Nimblegen (Madison, WI). For details, see manufacturer's web site www.nimblegen.com.
Hybridization protocol
Labeling and DNA hybridization onto a human hg17 custom designed oligonucleotide array (50mers) was carried out by Roche/NimbleGen (Madison, WI). The 2006-10-26_HG17_HELP_Promoter array covers 25,626 HpaII amplifiable fragments (HAF). EpiTyper by MassArray (Sequenom, CA) was used to confirm methylation of selected loci. Uniformity of hybridization was evaluated by adapting a published algorithm for the NimbleGen platform. Hybridizations with strong regional artifacts were discarded and repeated. Normalized signal intensities from each array were compared with a 20% trimmed mean of signal intensities across all arrays in that experiment. Arrays with significant intensity bias that could not be explained by the biology of the sample were excluded. (Sequenom, CA) was used to confirm methylation of selected loci as previously described.
Scan protocol
See manufacturer's web site www.nimblegen.com.
Description
HC5_LT_HSC_268624
Data processing
Signal intensities at each HAF were calculated as 25% trimmed mean of their component probe-level signal intensities. Any fragments found within the level of background MspI signal intensity (equaling 2.5 mean-absolute-difference, MAD) above the median of random probe signals were regarded failed. These failed loci represent the population of fragments that did not amplify by PCR. Loci were designated methylated when the level of HpaII signal intensity was indistinguishable from background as described for MspI. Fragments successfully amplified by PCR, i.e. distinguishable above background, were subjected to normalization. For this, an intra-array quantile approach was used: HpaII/MspI ratios are aligned across density-dependent sliding windows of fragment size-sorted data. The log2(HpaII/MspI) was used as a representative for methylation and analyzed as a continuous variable. If the centered log2(HpaII/MspI) ratio was <0, the corresponding fragment was considered methylated. It was considered hypomethylated in cases where log2(HpaII/MspI) was >0.
