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Sample GSM3040884 Query DataSets for GSM3040884
Status Public on Sep 06, 2018
Title MethylC-seq_P7_Kidney_EC_R2
Sample type SRA
 
Source name P7 GFP-positive sorted cells from Kidney (vascular ECs)
Organism Mus musculus
Characteristics strain/genotype: Tie2-GFP
age: P7
Sex: Male
tissue: Kidney
cell-type: Vascular endothelial cells
Growth protocol Adult mice were housed in our animal facility with 12 h light/dark cycles and food ab libitum. Animals were used for analysis in accordance with protocols approved by the Institutional Animal Care and Use Committee
Extracted molecule genomic DNA
Extraction protocol DNA for MethylC-seq was prepared from GFP-positive FACS-sorted cells using the DNeasy Blood and Tissue kit (69504, QIAGEN).
DNA methylome libraries were prepared using a modified snmC-seq protocol adapted for bulk DNA samples (Luo et al., 2017). 20 ng of purified genomic DNA with 0.5% unmethylated lambda DNA spike-in (D1521, Promega) was bisulfite converted using the EZ DNA Methylation-Direct™ Kit (D5021, Zymo) following the product manual and was eluted in 10 µl M-Elution buffer. 9 µl converted DNA was mixed with 1 µl P5L_random oligo (5 µM, /5SpC3/TTCCCTACACGACGCTCTTCCGATCTNNNNNNNNN, IDT) followed by heat denaturing at 95°C for 3 min using a thermocycler. The samples were chilled on ice for 2 min and mixed with 10 µl enzyme mix containing 2 µl of Blue Buffer (B0110, Enzymatics), 1 µl of 10 mM dNTP (N0447L, NEB), 1 µl of Klenow exo- (50 U/µl, P7010-HC-L, Enzymatics) and 6 µl H2O. The samples were incubated using a thermocycler with the following program: 4°C for 5 min, ramp up to 25°C at 0.1°C/sec, 25°C for 5 min, ramp up to 37°C at 0.1°C/sec, 37°C for 60 min, 4°C. A 3 µl enzyme mix containing 2 µl of Exonuclease 1 (20U/µl, X8010L, Enzymatics) and 1 µl of Shrimp Alkaline Phosphatase (rSAP, M0371L, NEB) was added to the samples. The samples were incubated at 37°C for 30 min using a thermocycler. Samples were cleaned-up using 0.8x SPRI beads and eluted with 10 µl M-Elution buffer. Eluted samples were heated at 95°C for 3 min using a thermocycler and were chilled on ice for 2 min. Samples were mixed with 10.5 ul Adaptase master mix containing 2 ul Buffer G1, 2 ul Regent G2, 1.25 ul Reagent G3, 0.5 ul Enzyme G4, 0.5 ul Enzyme G5 and 4.25 ul M-Elution buffer (Accel-NGS Adaptase Module for Single Cell Methyl-Seq Library Preparation, 33096, Swift Biosciences). Samples were incubated at 37°C for 30 min, 95°C for 2 min and 4°C using a thermocycler. 1 µl 30 µM P5 indexing primer and 5 µl 10 µM P7 indexing primer and 25 µl KAPA HiFi HotStart ReadyMix, (KK2602, KAPA BIOSYSTEMS) were added into each sample followed by thermocycling using the following program - 95°C for 2 min, 98°C for 30 sec, 10 cycles of (98°C for 15 sec, 64°C for 30 sec, 72°C for 2min), 72°C for 5 min and then 4°C. PCR products were cleaned up using 0.8x SPRI beads twice. Methylome libraries were sequenced using an Illumina HiSeq 4000 instrument with 5% PhiX DNA spike-in.
 
Library strategy Bisulfite-Seq
Library source genomic
Library selection RANDOM
Instrument model Illumina HiSeq 4000
 
Description MethylC-seq.bed.tar
Data processing Adaptor sequences were trimmed from sequencing reads using Cutadapt 1.11 with parameters -f fastq -q 20 -m 50 -a AGATCGGAAGAGCACACGTCTGAAC -A AGATCGGAAGAGCGTCGTGTAGGGA. We further trimmed 10 bp from both 5’- and 3’- ends of both R1 and R2 reads with parameters -f fastq -u 10 -u -10 -m 30. Trimmed R1 and R2 reads were mapped to the mm10 reference genome as single-end reads using bismark 0.14.4 with parameter --bowtie2. --pbat option was used for mapping R1 reads. Mapped reads were sorted using Samtools 1.3 followed by removing duplicate reads using Picard 1.141 MarkDuplicates with parameter REMOVE_DUPLICATES=true. Reads were further filtered by MAPQ > 20 using samtools view with parameter -bhq20. Methylation level at each cytosine was summarized using call_methylated_sites from Methylpy package (https://github.com/yupenghe/methylpy/).
genome build: mm10
Supplementary_files_format_and_content: bed files (in tar archive on series record) and tar of chromosome-specific files with read counts.
 
Submission date Mar 14, 2018
Last update date Apr 09, 2019
Contact name Mark F Sabbagh
E-mail(s) msabbag2@jhmi.edu
Organization name Johns Hopkins University School of Medicine
Department Molecular Biology and Genetics
Lab Jeremy Nathans
Street address 725 North Wolfe Street
City Baltimore
State/province MD
ZIP/Postal code 21205
Country USA
 
Platform ID GPL21103
Series (1)
GSE111839 Transcriptional and Epigenomic Landscapes of CNS and non-CNS Vascular Endothelial Cells
Relations
BioSample SAMN08712801
SRA SRX3791757

Supplementary file Size Download File type/resource
GSM3040884_MethylC-seq_P7_Kidney_EC_R2.bw 170.3 Mb (ftp)(http) BW
GSM3040884_allc_MSD15.tar.gz 4.0 Gb (ftp)(http) TAR
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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