title: epiRIL218_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=448470_635_A02.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=449787_532_A02.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL218_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: epiRIL315_Col_BC1-S7_IP vs WT_Col_INPUT

status: Public on Sep 03 2012

type: genomic

city: PARIS

first: Vincent

last: Colot

address: 46 rue d'Ulm

country: France

institute: IBENS - CNRS UMR8197 - INSERM U1024

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=388464_635_A01.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=388463_532_A01_inv.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

hyb_protocol: Co-hybridization was performed using the NimbleGen 3x720K array design and following manufacturer's instructions

scan_protocol: Data acquisition was performed according to Roche Nimblegen instructions.

data_processing: The rescaling of the MeDIP data was done using R computing software (version 2.10.1) as described in [1]. Signals were re-expressed as log2 ratios of IP and INPUT signal. We used the averaged Col-wt input signal as a common input for all samples. For each sample one extra dye-swap experiment was available except for the parents (3 and 2 dye-swapped replicates for Col-wt and Col-ddm1 respectively). The dye-swapped replicates were in all cases averaged to account for possible dye bias. We standardized the overall signal distribution by expressing it in terms of the standard deviation of the intron probe signal distribution (probes that match with intron sequences). This has the effect of placing the mean of the intron probe signal at zero and rescaling the values as standard deviation values. We applied a HMM model to the data to call methylated, intermediate methylated and unmethylated probes, as described in [1]. We used the signal distribution of the probes that match with intron sequences to obtain the emission probability of the unmethylated hidden state (intron sequences are in general unmethylated). The signal distribution of the introns was approximated with a mixture of 30 Gaussian distributions (estimation was carried with the EM algorithm [2], which was implemented in R). The emission probability of the methylated hidden state was a Gaussian with mean fixed at the 99th quantile of the emission probability of the unmethylated state (i.e. the signal distribution for introns) and the variance of the distribution was estimated (Baum-Welch algorithm [3,4]). The emission probability for the intermediate methylated probes was a Gaussian with mean fixed between the mean of the emission probability of the unmethylated hidden state (i.e. the intron distribution) and the mean of the emission probability of the methylated hidden state, and variance equal to the variance of the emission probability of the methylated hidden state. [1] Cortijo S, Wardenaar R, Colome-Tatche M, Johannes F and Colot V (2012) Genome-wide analysis of DNA methylation in Arabidopsis using MeDIP-chip. In: McKeown P and Spillane C (Eds) Plant Epigenome: Understanding and Analysis, Humana Press/Springer: New York. [2] McLachlan and Peel (2000), Finite Mixture Models, John Wiley and Sons, ISBN: 0471006262, 9780471006268, New York. [3] Baum LE, T. Petrie, G. Soules, and N. Weiss (1970) A maximization technique occurring in the statistical analysis of probabilistic functions of Markov chains, Ann. Math. Stat., 41, 164–171. [4] Rabiner LR (1989) A tutorial on hidden Markov models and selected applications in speech recognition. Proceedings of the IEEE 77, 257-286

label: Cy3, Cy5

molecule: genomic DNA

organism: Arabidopsis thaliana

label_protocol: INPUT DNA was amplified using the Sigma GenomePlex Complete Whole Genome Amplification (WGA) Kit according to manufacturer's instructions. Cy3 and Cy5 labeling were performed using the Nimblegen Dual color DNA labeling kit (Roche NimbleGen) according to manufacturer's instructions.

growth_protocol: Replicates 1 and 2 : 12 days-old seedlings grown in liquid media, 16 hours photoperiod. Replicate 3 : Aerial parts of 21 day-old plants grown in soil, 16 hours photoperiod.

extract_protocol: WT_Col_INPUT: DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor.

treatment_protocol: no treatment

tag: ecotype

tag: age

value: 12 days for replicates 1 and 2, 21 days for replicate 3

tag: developmental stage

value: Seedlings for replicates 1 and 2, aerial part for replicate 3

tag: antibody

value: none (input)

label: Cy5, Cy3

label_protocol: IP DNA was amplified using the Sigma GenomePlex Complete Whole Genome Amplification (WGA) Kit according to manufacturer's instructions. Cy3 and Cy5 labeling were performed using the Nimblegen Dual color DNA labeling kit (Roche NimbleGen) according to manufacturer's instructions.

extract_protocol: epiRIL315_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.

value: 21 days

value: aerial parts

tag: vendor

value: Diagenode

tag: catalog#


title: epiRIL202_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=388470_635_A01.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=388470_532_A02.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

growth_protocol: Aerial parts of 21 day-old plants grown in soil, 16 hours photoperiod.

extract_protocol: epiRIL202_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: epiRIL172_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=388447_635_A03.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=383565_532_A03.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL172_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: epiRIL166_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=430073_635_A02.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=430069_532_A02.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL166_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: epiRIL169_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=429866_635_A03.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=429869_532_A03.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL169_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: epiRIL164_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=429795_635_A02.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=429786_532_A02.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL164_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: epiRIL147_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=449611_635_A02.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=449193_532_A01.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL147_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: epiRIL170_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=429872_635_A02.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=429817_532_A02.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL170_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: epiRIL159_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=429782_635_A02.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=429761_532_A02.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL159_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: epiRIL150_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=465394_635_A03.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=465397_532_A03.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL150_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: epiRIL118_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=388487_635_A01.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=388484_532_A01.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL118_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: epiRIL114_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=465394-2_635_A03.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=465399-2_532_A03.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL114_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: epiRIL148_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=448079_635_A01.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=448069_532_A01.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL148_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: epiRIL122_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=429953_635_A02.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=429952_532_A02.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL122_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: epiRIL112_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=429797_635_A03.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=429797_532_A02.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL112_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: epiRIL101_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=429782_635_A01.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=429785_532_A01.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL101_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: epiRIL144_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=465372_635_A02.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=465406_532_A02.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: WT_Col_INPUT: DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor.

extract_protocol: epiRIL144_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: epiRIL070_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=359196_635_A01.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=388777_532_A01.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL070_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: epiRIL137_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=429872_635_A01.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=429817_532_A01.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL137_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: epiRIL098_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=429866_635_A02.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=429869_532_A02.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL098_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: epiRIL094_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=449611_635_A01.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=449193_532_A02.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL094_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: epiRIL073_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=429887_635_A01.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=429884_532_A01.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL073_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: epiRIL071_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=449881_635_A01.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=449879_532_A01.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL071_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: epiRIL099_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=448301_635_A01.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=448300_532_A01.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL099_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: epiRIL093_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=429887_635_A02.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=429884_532_A02.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL093_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: epiRIL095_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=448459_635_A01.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=448304_532_A01.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL095_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: epiRIL069_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=511503-2_635_A01.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=511503-2_532_A02.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL069_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: epiRIL108_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=449878_635_A01.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=449852_532_A03.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL108_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: epiRIL244_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=429932_635_A01.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=429889_532_A01.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL244_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: PBC_23 tumor27

type: RNA

city: Amsterdam

first: Daoud

last: Sie

address: De Boelelaan 1117

department: Pathology

country: Netherlands

institute: Vrije Universiteit Medical Center

description:
Sample.23.cy5
raw data file: MAL_251485027359_S01_GE2-v5_95_Feb07_1_3.txt

hyb_protocol: Hybridization was performed on a human GE 4x44K formatted expression array spotted with 60-mer oligonucleotides representing over 43,000 well-characterized coding and non-coding sequences (G4112F, Agilent Technologies, Amstelveen, The Netherlands).  Input: 825 ng RNA

scan_protocol: Scanning was performed using a microarray scanner G2505B (Agilent Technologies). Feature Extraction v9.5 using the manufacturers protocols (Agilent Technologies).

data_processing:
Preprocessing of the gene expression data comprised of RMA background correction, loess within-array normalization and A-quantile between-array normalization was performed in R. Single channel data were used to accommodate the unbalanced design. Ch1=cy3, ch2=cy5
The moderated t-test implemented in Limma, employing empirical Bayes estimation of the variance, was used to evaluate the difference in gene expression between the two groups. Sample origin and CyDye were taken along as possible confounders. The Benjamini-Hochberg procedure was applied to the raw p-values to control the False Discovery Rate (FDR).Tobacco use was classified as ‘none’ when the individual did not smoke for a period of at least half a year before sampling. Alcohol use was scored as ‘none’, when the individual drank no alcohol or if one had abstained from alcohol for more than half a year. Analysis was performed with and without correction for tobacco and alcohol consumption.
There were 28 patients with head and neck squamous cell carcinoma (HNSCC) with samples labeleld 'tumor''. There were 11 individuals without cancer that served as control, with samples labeled 'control'. Each patient and control has an unique number.
Although the hybridizations were dual channel (two samples per array), the data were analyzed as single channel (each sample independently).
Data for several Samples was not used in the analysis and are omitted from the processed data and Sample list: Sample 11 cy3, Sample 16 cy5, Sample 17 cy3, Sample 18 cy3, Sample 19 cy5, Sample 20 cy3, Sample 21 cy5, Sample 22 cy5, Sample 23 cy3, Sample 24 cy5, Sample 25 cy3

molecule: total RNA

organism: Homo sapiens

source_name: Nucleated peripheral blood cells

label_protocol: cDNA synthesis and labeling were performed according to the recommendations of the manufacturer (Agilent Technologies, Amstelveen, The Netherlands), including quality controls (QCs)

extract_protocol: These PBC were lysed with RNA-bee (Campro Scientific bv., Veenendaal, the Netherlands)  and RNA was extracted according to the protocol of the manufacturer.

treatment_protocol: Blood collection was performed when the patient was under general anesthesia, before the surgical procedure had started. Blood samples by venipuncture were collected in heparin tubes. After lysis of the erythrocytes by osmotic shock, the nuclear cells were washed with Hanks buffered salt solution.

tag: cancer or control with number

tag: current tobacco consumption

value: no

tag: current alchol consumption

value: yes


title: epiRIL092_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=448470_635_A01.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=449787_532_A01.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL092_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: PBC_25 tumor28

description:
Sample.25.cy5
raw data file: MAL_251485027360_S01_GE2-v5_95_Feb07_1_2.txt


title: epiRIL064_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=429953_635_A01.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=429952_532_A01.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL064_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: epiRIL054_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=388451_635_A01.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=388657_532_A01.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: WT_Col_INPUT: DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor.

extract_protocol: epiRIL054_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: epiRIL062_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=465372_635_A01.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=465406_532_A01.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL062_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: PBC_21 tumor13

description:
Sample.21.cy3
raw data file: MAL_251485027358_S01_GE2-v5_95_Feb07_1_4.txt


title: PBC_22 tumor14

description:
Sample.22.cy3
raw data file: MAL_251485027359_S01_GE2-v5_95_Feb07_1_2.txt


title: PBC_24 tumor15

description:
Sample.24.cy3
raw data file: MAL_251485027360_S01_GE2-v5_95_Feb07_1_1.txt


title: epiRIL055_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=388451_635_A02.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=388657_532_A02.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL055_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: epiRIL060_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=429866_635_A01.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=429869_532_A01.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL060_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: epiRIL046_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=448352_635_A02.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=448316_532_A02.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL046_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: PBC_16 tumor11

description:
Sample.16.cy3
raw data file: MAL_251485027356_S01_GE2-v5_95_Feb07_1_3.txt


title: PBC_19 tumor12

description:
Sample.19.cy3
raw data file: MAL_251485027357_S01_GE2-v5_95_Feb07_1_3.txt

value: unknown


title: PBC_15 control11

description:
Sample.15.cy5
raw data file: US22502676_251485060061_S01_GE2-v5_95_Feb07_1_1.txt


title: PBC_17 tumor24

description:
Sample.17.cy5
raw data file: MAL_251485027356_S01_GE2-v5_95_Feb07_1_4.txt


title: PBC_18 tumor25

description:
Sample.18.cy5
raw data file: MAL_251485027357_S01_GE2-v5_95_Feb07_1_2.txt


title: PBC_14 tumor23

description:
Sample.14.cy5
raw data file: US22502676_251485059325_S01_GE2-v5_95_Feb07_1_4.txt


title: PBC_20 tumor26

description:
Sample.20.cy5
raw data file: MAL_251485027358_S01_GE2-v5_95_Feb07_1_1.txt


title: PBC_15 tumor10

description:
Sample.15.cy3
raw data file: US22502676_251485060061_S01_GE2-v5_95_Feb07_1_1.txt


title: PBC_14 tumor9

description:
Sample.14.cy3
raw data file: US22502676_251485059325_S01_GE2-v5_95_Feb07_1_4.txt


title: epiRIL053_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=429894_635_A01.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=429893_532_A01.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL053_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: epiRIL036_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=429795_635_A01.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=429786_532_A01.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL036_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: PBC_11 tumor20

description:
Sample.11.cy5
raw data file: US22502676_251485059325_S01_GE2-v5_95_Feb07_1_1.txt


title: epiRIL014_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=359115_635_A03.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=358406_532_A03.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL014_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: PBC_13 tumor22

description:
Sample.13.cy5
raw data file: US22502676_251485059325_S01_GE2-v5_95_Feb07_1_3.txt


title: PBC_12 tumor7

description:
Sample.12.cy3
raw data file: US22502676_251485059325_S01_GE2-v5_95_Feb07_1_2.txt


title: PBC_9 control10

description:
Sample.9.cy5
raw data file: US22502676_251485059324_S01_GE2-v5_95_Feb07_1_2.txt


title: PBC_10 control4

description:
Sample.10.cy3
raw data file: US22502676_251485059324_S01_GE2-v5_95_Feb07_1_3.txt


title: PBC_12 tumor21

description:
Sample.12.cy5
raw data file: US22502676_251485059325_S01_GE2-v5_95_Feb07_1_2.txt


title: PBC_13 tumor8

description:
Sample.13.cy3
raw data file: US22502676_251485059325_S01_GE2-v5_95_Feb07_1_3.txt


title: PBC_10 tumor19

description:
Sample.10.cy5
raw data file: US22502676_251485059324_S01_GE2-v5_95_Feb07_1_3.txt


title: A197_11_SI3_YLF

city: Wageningen

first: Guido

last: Hooiveld

address: HELIX, Stippeneng 4

department: Div. Human Nutrition & Health

institute: Wageningen University

hyb_protocol: Hybridization of 5.5μg labelled cDNA was done overnight for 17 hours, at 60 rpm, at 45ºC in a Hybridization Oven 640 (Affymetrix). The protocol was conducted as described in the Affymetrix Whole Transcript (WT) Sense Target Labeling Assay Manual, chapter 5 (P/N 701880, revision 5). Washing and staining of the arrays were done on an Affymetrix 450 fluidics station using the protocol FS450_0001, as described in the Affymetrix Whole Transcript (WT) Sense Target Labeling Assay Manual, chapter 5 (P/N 701880, revision 5).

scan_protocol: Arrays were scanned on an Affymetrix 3000 7G scanner, as described in the Genechip Expression Analysis Technical Manual, section 2 (Eukaryotic Sample and Array Processing), chapter 2 (Eukaryotic Arrays: Washing, Staining and Scanning (P/N 701028, revision 5).

data_processing: Expression estimates were calculated applying the RMA algorithm in the Bioconductor library 'Oligo' (v1.20.4).

label: biotin

organism: Mus musculus

source_name: scraping of the distal segment of the small intestine after 2 weeks low-fat diet, pooled sample of 6 young mice

label_protocol: The Affymetrix GeneChip WT Sense Target Labeling and Control Reagents kit (P/N 900652) was used for the preparation of labelled cDNA from 100ng of total RNA without rRNA reduction. A detailed description can be found in the User Manual, Chapter 3 (P/N 701880, revision 5).

growth_protocol: Male C57BL/6J mice of 12 weeks and 20 months of age were purchased from JANVIER SAS, Le Genest Saint Isle, France, and  housed individually in a light and temperature-controlled animal facility of Wageningen University (12:12 h light/dark cycle, 20±2 °C). Mice were fed standard lab chow (RMH-B; Arie Blok BV, Woerden, the Netherlands) and had free access to feed and water.

extract_protocol: Total RNA was prepared from intestinal scrapings using TRIzol reagent, whereafter purified total RNA was isolated using Qiagen RNEasy columns. RNA integrity was checked on chip analysis (Agilent 2100 bioanalyzer, Agilent Technologies, Amsterdam, the Netherlands) according to the manufacturer's instructions. RNA was judged as suitable for array hybridization only if samples exhibited intact bands corresponding to the 18S and 28S ribosomal RNA subunits, and displayed no chromosomal peaks or RNA degradation products (RNA Integrity Number > 8.0). For array analysis, RNA of 6-8 mice per group was subsequently pooled at a concentration of 500 ng/mouse.

treatment_protocol: After been fed for 1 week the lab chow, mice were given a semi-synthetic low-fat (10%) diet for 2 weeks. Subsequently, both age groups were split into two subgroups one of which received the low-fat diet for two more weeks while the other group received a high-fat (45%) diet for two weeks. Both diets were based on Research Diets’ formulas D12450B/D12451, with adaptations regarding type of fat (palm oil instead of lard) and carbohydrates. After this intervention period, mice were killed in the postprandial state and the small intestine was removed.

tag: strain

tag: gender

value: male

value: 4 months

tag: tissue

value: distal part small intestine


title: PBC_7 tumor18

description:
Sample.7.cy5
raw data file: US22502676_251485059323_S01_GE2-v5_95_Feb07_1_4.txt


title: PBC_8 control9

description:
Sample.8.cy5
raw data file: US22502676_251485059324_S01_GE2-v5_95_Feb07_1_1.txt


title: epiRIL020_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=429888_635_A02.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=429771_532_A02.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL020_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: A197_12_SI3_YHF

source_name: scraping of the distal segment of the small intestine after 2 weeks high-fat diet, pooled sample of 6 young mice


title: PBC_9 tumor6

description:
Sample.9.cy3
raw data file: US22502676_251485059324_S01_GE2-v5_95_Feb07_1_2.txt


title: epiRIL024_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=430073-2_635_A03.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=430069-2_532_A03.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL024_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: epiRIL018_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=429888_635_A01.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=429771_532_A01.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL018_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: epiRIL008_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=465394-2_635_A02.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=465404-2_532_A03.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL008_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: A197_07_SI2_YLF

source_name: scraping of the middle segment of the small intestine after 2 weeks low-fat diet, pooled sample of 6 young mice

value: middle part small intestine


title: PBC_5 tumor3

description:
Sample.5.cy3
raw data file: US22502676_251485059323_S01_GE2-v5_95_Feb07_1_1.txt

extract_protocol: These PBC were lysed with RNA-bee (Campro Scientific bv., Veenendaal, the Netherlands)  and RNA was extracted according to the protocol of the manufacturer.

value: y


title: A197_10_SI3_OHF

source_name: scraping of the distal segment of the small intestine after 2 weeks high-fat diet, pooled sample of 8 old mice

value: 21 months


title: PBC_7 control3

description:
Sample.7.cy3
raw data file: US22502676_251485059323_S01_GE2-v5_95_Feb07_1_4.txt


title: A197_06_SI2_OHF

source_name: scraping of the middle segment of the small intestine after 2 weeks high-fat diet, pooled sample of 8 old mice


title: A197_08_SI2_YHF

source_name: scraping of the middle segment of the small intestine after 2 weeks high-fat diet, pooled sample of 6 young mice


title: PBC_6 tumor4

description:
Sample.6.cy3
raw data file: US22502676_251485059323_S01_GE2-v5_95_Feb07_1_2.txt


title: A197_05_SI2_OLF

source_name: scraping of the middle segment of the small intestine after 2 weeks low-fat diet, pooled sample of 8 old mice


title: A197_03_SI1_YLF

source_name: scraping of the proximal segment of the small intestine after 2 weeks low-fat diet, pooled sample of 6 young mice

value: proximal part small intestine


title: epiRIL011_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=511503_635_A01.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=511527_532_A01.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: epiRIL011_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.


title: PBC_4 tumor17

description:
Sample.4.cy5
raw data file: US22502676_251485056689_S01_GE2-v5_95_Feb07_1_4.txt


title: A197_04_SI1_YHF

source_name: scraping of the proximal segment of the small intestine after 2 weeks high-fat diet, pooled sample of 6 young mice


title: PBC_8 tumor5

description:
Sample.8.cy3
raw data file: US22502676_251485059324_S01_GE2-v5_95_Feb07_1_1.txt


title: PBC_5 control7

description:
Sample.5.cy5
raw data file: US22502676_251485059323_S01_GE2-v5_95_Feb07_1_1.txt


title: PBC_3 tumor16

description:
Sample.3.cy5
raw data file: US22502676_251485056689_S01_GE2-v5_95_Feb07_1_3.txt


title: PBC_2 tumor2

description:
Sample.2.cy3
raw data file: US22502676_251485056689_S01_GE2-v5_95_Feb07_1_2.txt


title: PBC_2 control6

description:
Sample.2.cy5
raw data file: US22502676_251485056689_S01_GE2-v5_95_Feb07_1_2.txt


title: PBC_4 control2

description:
Sample.4.cy3
raw data file: US22502676_251485056689_S01_GE2-v5_95_Feb07_1_4.txt


title: A200_04_colonscr_YHF

source_name: scraping of the colon after 2 weeks high-fat diet, pooled sample of 6 young mice

extract_protocol: Total RNA was prepared from colonic scrapings using TRIzol reagent, whereafter purified total RNA was isolated using Qiagen RNEasy columns. RNA integrity was checked on chip analysis (Agilent 2100 bioanalyzer, Agilent Technologies, Amsterdam, the Netherlands) according to the manufacturer's instructions. RNA was judged as suitable for array hybridization only if samples exhibited intact bands corresponding to the 18S and 28S ribosomal RNA subunits, and displayed no chromosomal peaks or RNA degradation products (RNA Integrity Number > 8.0). For array analysis, RNA of 6-8 mice per group was subsequently pooled at a concentration of 500 ng/mouse.

treatment_protocol: After been fed for 1 week the lab chow, mice were given a semi-synthetic low-fat (10%) diet for 2 weeks. Subsequently, both age groups were split into two subgroups one of which received the low-fat diet for two more weeks while the other group received a high-fat (45%) diet for two weeks. Both diets were based on Research Diets’ formulas D12450B/D12451, with adaptations regarding type of fat (palm oil instead of lard) and carbohydrates. After this intervention period, mice were killed in the postprandial state and the colon was removed.

value: colon


title: PBC_6 control8

description:
Sample.6.cy5
raw data file: US22502676_251485059323_S01_GE2-v5_95_Feb07_1_2.txt


title: PBC_3 control1

description:
Sample.3.cy3
raw data file: US22502676_251485056689_S01_GE2-v5_95_Feb07_1_3.txt


title: A197_01_SI1_OLF

source_name: scraping of the proximal segment of the small intestine after 2 weeks on low-fat diet, pooled sample of 8 old mice


title: A200_03_colonscr_YLF

source_name: scraping of the colon after 2 weeks low-fat diet, pooled sample of 6 young mice


title: A200_02_colonscr_OHF

source_name: scraping of the colon after 2 weeks high-fat diet, pooled sample of 8 old mice


title: PBC_1 control5

description:
Sample.1.cy5
raw data file: US22502676_251485056689_S01_GE2-v5_95_Feb07_1_1.txt


title: A197_02_SI1_OHF

source_name: scraping of the proximal segment of the small intestine after 2 weeks high-fat diet, pooled sample of 8 old mice


title: Transcriptome epiRIL202_Col vs WT_Col [ds1 and ds2]

description:
Transcriptome analysis in dye-swap.
1st hybridization raw data:
epiRIL202=448079-2_635_A02.pair - WT=448079-2_532_A02.pair
swap raw data:
epiRIL202=388467-2_532_A02.pair - WT=388467-2_635_A02.pair

hyb_protocol: Co-hybridization was performed using the NimbleGen 3x720K array design and following manufacturer’s instructions

data_processing: Hybridization data were normalized using an ANOVA model to remove technical biases. Data were averaged on the dye-swap to remove tile-specific dye biases.

label_protocol: Cy3 and Cy5 labeling were performed using the Nimblegen Dual color DNA labeling kit (Roche NimbleGen) according to manufacturer’s instructions, amplification=yes, 1 ug of total RNA were amplified with one round of in vitro transcription 10h at 37°C using the MessageAmp™ II aRNA Amplification Kit (Ambion). Double stranded cDNA synthesis was then performed on 2 ug of aRNA using the SuperScript® Double-Stranded cDNA Synthesis Kit (Invitrogen).

growth_protocol: Seedlings 10 day-old plants grown in liquid 1/2MS media, 16 hours photoperiod.

extract_protocol: WT_Col:10ug. Total RNA was extracted using Rneasy Plant Minikit (Qiagen) according to the supplier’s instructions.

tag: genotype/variation

value: WT

value: 10 days

value: whole seedlings

extract_protocol: epiRIL202_Col:10ug. Total RNA was extracted using Rneasy Plant Minikit (Qiagen) according to the supplier’s instructions.


title: Transcriptome ddm1_Col vs WT_Col [ds1 and ds2]

description:
Transcriptome analysis in dye-swap.
1st hybridization raw data:
ddm1=388467-2_635_A01.pair - WT=448079-2_532_A01.pair
swap raw data:
ddm1=388467-2_532_A01.pair - WT=448079-2_635_A01.pair

extract_protocol: ddm1_Col:10ug. Total RNA was extracted using Rneasy Plant Minikit (Qiagen) according to the supplier’s instructions.


title: epiRIL052_Col_BC1-S7_IP vs WT_Col_INPUT

description:
Methylome analysis in dye-swap. 1st hybridization raw data:  IP=430073_635_A01.pair - INPUT=INPUTwt_532_3rep.pair ; swap raw data: IP=430069_532_A01.pair - INPUT=INPUTwt_635_3rep.pair
Raw files INPUTwt_532_3rep.pair, INPUTwt_635_3rep.pair are linked as supplementary files on the Series record.

extract_protocol: WT_Col_INPUT: DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor.

value: 12 days for replicates 1 and 2, 21 days for replicate 3

value: Seedlings for replicates 1 and 2, aerial part for replicate 3

value: none (input)

extract_protocol: epiRIL052_Col_BC1-S7_IP. DNA was extracted using the Qiagen MaxiPrep kit (Qiagen) and sonicated using a Diagenode Bioruptor. Immunoprecipitation was done on 1.5µg of DNA (IP fraction) and 150ng serves as INPUT DNA. First, IP fraction was denaturated by incubating 10 minutes at 95°C and 2 minutes on ice in 600µl of buffer 1 (10 mM Tris HCl pH 7.5, 500 mM NaCl, 1 mM EDTA). Immunoprecipitation was performed by adding 5µg of 5mC antibody (Diagenode, cat n° MAb-006-500) to denatured IP fraction and incubating overnight at 4°C with gentle agitation. Forty microliters of washed M280 Dynabeads (Invitrogen) were added to the IP fraction and incubated at 4°C for 4 hr with gentle agitation. Supernatant was collected in a new tube (Supernatant fraction). Three hundred microliters of buffer 1 were added to IP tube and incubated 10 min at room temperature with gentle agitation. This first wash was added to Supernatant fraction. Then IP fraction was washed three times by gentle mixing for 10 min at room temperature with 600 ml of buffer 1. To elute, IP fraction was incubated 1 hour at 42°C with 300µl of buffer 2 (30mM Tris HCl pH: 8.0) and 7µl of Proteinase K (NEB, 20µg/µl). DNA from the Supernatant an IP fractions was recovered by phenol-chloroform extraction and ethanol precipitation.

value: 21 days