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Sample GSM2220281 Query DataSets for GSM2220281
Status Public on Jun 20, 2017
Title GM_11 scNOMe-seq Sample
Sample type SRA
 
Source name lymphoblast cell line
Organism Homo sapiens
Characteristics cell line: GM12878
enzyme: GpC Methyltransferase M.CviPl (NEB)
Biomaterial provider Coriell Cell Repositories https://catalog.coriell.org/0/Sections/Search/Sample_Detail.aspx?Ref=GM12878
Growth protocol GM12878 and K562 cells were obtained from Coriell and ATCC, respectively. GM12878 were grown in RPMI medium 1640 (Gibco), supplemented with 2mM L-Glutamine (Gibco), and Penicilin and Streptavidin (Pen Strep, Gibco), and 15% fetal bovine serum (FBS, Gibco). K562 were grown in RPMI medium 1640 of the same composition but with 10% FBS.
Extracted molecule genomic DNA
Extraction protocol Between 2 M and 5 M cells were collected by centrifuging the cell suspension for 5 min at 500x g. Cells were washed once with 1x PBS, resuspended in 1 ml lysis buffer (10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2) and incubated for 10 min on ice. IGEPAL CA-630 (Sigma) was added to a final concentration of 0.025% and cell suspension was transferred to a 2 ml Dounce homogenizer. Nuclei were released by 15 strokes with the pestle. Success of lysis was confirmed by inspection under a light microscope. Nuclei were pelleted at at 4C and washed twice with cold lysis buffer without detergent. One million nuclei were resupended in reaction buffer to yield 100 ul with a final concentration of 1x GpC MTase buffer (NEB), 0.32 mM S-Adenosylmethionine (SAM) (NEB). 50 ul of GpC methyltransferase (4U/ul)) from M.CviPl (NEB)were added. The suspension was carefully mixed before incubation for 8 min at 37C after which another 25 ul of enzyme and 0.7 ul of 32mM SAM were added for an additional 8 min incubation at 37C. To avoid disruption of nuclei incubation was stopped by adding 750 ul of 1x PBS and pelleting the nuclei at 800 xg. Supernatant was removed and nuclei were resuspended in 500 ul 1x PBS containing Hoechst 33342 DNA dye (NucBlue Live reagent, Hoechst). Nuclei were kept on ice until sorting. Nuclei were sorted at the Flow Cytometry core at the University of Chicago on a BD FACSAria or BD FACSAria Fusio equipped with a 96-well-plate holder. To obtain individual and intact nuclei gates were set to exclude aggregates and debris, and to include cells with DNA content corresponding to cells in G1 phase of the cell cycle to maintain similar DNA content per cell and to remove potential heterogeneity attributable to cell cycle. Cells were sorted into individual wells pre-filled wth 19 ul of 1x M-Digestion buffer (EZ DNA Methylation Direct Kit, Zymo Research) containing 1mg/ml Proteinase K. Following collection, the plates were briefly spun to collect droplets that might formed during handling. Nuclei were lysed by incubating the samples at 50C for 20 min in a PCR cycler. DNA was subjected to bisulfite conversion by adding 130 ul of freshly prepared CT Conversion reagent (EZ DNA Methylation Direct Kit, Zymo) to the lysed nuclei. Conversion was performed by denaturing the DNA at 98C for 8 min followed by 3.5 hrs incubation at 65C. DNA isolation was performed using the EZ DNA Methylation Direct Kit (Zymo Research) following the manufacturer’s instruction with the modification that the DNA was eluted in only 8 ul of elution buffer.
Libraries were prepared using the Pico Methyl-seq Library prep Kit (Zymo Research) following the manufacturer’s instruction for low input samples. Specifically, the random primers were diluted 1:2 before the initial pre-amplification step and the first amplification extended to a total of 10 amplification cycles. Libraries were amplified with barcoded primers allowing for multiplexing. The sequences can be found in supplementary material, primers were ordered from IDT. The purification of amplified libraries was performed using Ampure beads, using a 1:1 ratio of bedas and libraries. Concentration and size distribution of the final libraries was assessed on an Bioanalyzer (Agilent). Libraries with average fragment size above 150bp were pooled and sequenced.
 
Library strategy Bisulfite-Seq
Library source genomic
Library selection RANDOM
Instrument model Illumina HiSeq 4000
 
Description bisulfite converted DNA
Data processing reads were trimmed to remove low quality bases and 6 bp were clipped from the 5prime end of each read to avoid mismatches introduced by amplification, in the case of GM12878 cells 6 bp were clipped from either end. Only reads that remained longer than 20 bp were kept for further analyses. these processing steps were performed using trim_galore version 0.4.0 (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) with the following settings: trim_galore --quality 30 --phred33 --illumina --stringency 1 -e 0.1 --clip_R1 6 --gzip --length 20 --output_dir outdir Sample.fastq.gz
sequencing data was aligned to the in silico bisulfite converted human genome assembly hg38 using Bismark aligner v0.15 (Krueger, F., Kreck, B., Franke, A. & Andrews, S. R. DNA methylome analysis using short bisulfite sequencing data. Nat Meth 9, 145–151 (2012).) with default settings (bismark --fastq --prefix SamplePrefix --output_dir output_dir --non_directional --phred33-quals --score_min L,0,-0.2 --bowtie2 genome.index trimmed.fastq.gz). Reads were aligned in single end modus with each read of a paired-end read aligned separately
Coverage and methylation status of all cytosines was extracted using bismarkmethyl extract (bismark_methylation_extractor -s --ignore 6 --output outdir --cytosine_report --CX --genome_folder path_to_genome SampleAligned.bam).
Coverage files were used to extract the methylation status of cytosines specifically in GpC and CpG di-nucleotides using the coverage2cytosine script in Bismark. The resulting files contained the coverage and methylation information at individual GpC and CpG di-nucleotides. Cytosines in ambiguous GCG positions were removed from the coverage files. Regions covered by a read with more than 3 unconverted cytosines in non-CpG and non-GpC context were removed from further analyses as well.
Genome_build: hg38
Supplementary_files_format_and_content: GpC and CpG coverage files <chr><start><stop><%methylation><count methylated><total count> counts above 1 are a reflection of redundant reads that were not removed due to sequencing error or different length (after clipping)
 
Submission date Jun 29, 2016
Last update date May 15, 2019
Contact name Sebastian Pott
E-mail(s) spott@uchicago.edu
Organization name University of Chicago
Department Human Genetics
Street address 920 58th Street
City Chicago
State/province Il
ZIP/Postal code 60637
Country USA
 
Platform ID GPL20301
Series (1)
GSE83882 Simultaneous measurement of chromatin accessibility, DNA methylation, and nucleosome phasing in single cells
Relations
BioSample SAMN05326739
SRA SRX1885960

Supplementary file Size Download File type/resource
GSM2220281_GM_11_CpG_clean.cov.txt.gz 9.7 Mb (ftp)(http) TXT
GSM2220281_GM_11_GpC_clean.cov.txt.gz 48.2 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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