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Sample GSM3070829 Query DataSets for GSM3070829
Status Public on Sep 20, 2018
Title 180118_CEMBA_mm_P56_P63_4B_CEMBA180104_4B_1_CEMBA180104_4B_2_A12_AD006_indexed
Sample type SRA
 
Source name Mouse primary motor cortex
Organism Mus musculus
Characteristics Sex: Male
age: P56-P63
genome build: mm10
library strategy: snmC-seq2
Growth protocol Male C57Bl/6J mice were purchased from Jackson laboratories at 8 weeks of age and maintained in our facility for 48h before dissection. Animals were maintained in the Salk animal barrier facility on 12h dark-light cycles with food ad-libitum. Human brain specimens were obtained from University of Miami Brain Endowment Bank. snmC-seq with Shrimp Alkaline Phosphatase treatment (snmC-seq + SAP) was applied to frontal cortex (Brodmann Area 10, BA10) tissue of a deceased 58-year-old Caucasian male with PMI = 23.4. snmC-seq2 was applied to BA10 tissue of a deceased 25-year-old Caucasian male with PMI = 20.8.
Extracted molecule genomic DNA
Extraction protocol Bisulfite-converted samples were denatured at 95°C for 3 minutes, then placed on ice for 2 minutes. 5µL Random Priming master mix [1µL 10x Blue Buffer (Enzymatics B0110), 0.25µL Klenow Exo- (50U/µL, Enzymatics, P7010-HC-L), 0.5µL dNTP (10mM each, NEB N0447L), 3.25µL water] was added and incubated at 4°C for 5 min, 25°C for 5 min, and 37°C for 60 min followed by 4°C. 1.5µL enzyme mix containing Exonuclease 1 (20U/µL, Enzymatics X8010L) and rSAP (1U/µL, NEB M0371L) was added, then incubated at 37°C for 30 min followed by 4°C. 0.8x SPRI beads were added, mixed and incubated for 5 minutes at room temperature to allow DNA to bind. The beads were washed 3 times with 80% ethanol and eluted in 10µL EB buffer (Qiagen 19086). The plates were denatured again at 95°C for 3 minutes, and 10.5µL Adaptase master mix [2µL G1, 2µL G2, 1.25µL G3, 0.5µL G4 and 0.5µL G5 (Swift Biosciences 33096)] was added and incubated at 37°C for 30 minutes, then 95°C for 2 minutes. 25µL 2x KAPA HiFi HotStart ReadyMix (Kapa, KK2602) and 5µL custom indexing primer mix (6µM P5, 10µM P7) were added. The PCR was programmed as follows: 1) 95°C 2 min, 2) 98°C 30 sec, 3) 98°C 15 sec, 4) 64°C for 30 sec, 5) 72°C for 2 min, 6) 72°C 5 min, 7) 4°C hold. Repeat steps 3-5 for 15 total cycles. PCR reactions were cleaned with 0.8X SPRI beads for three rounds. Library concentration was determined with Qubit™ dsDNA BR Assay Kit (ThermoFisher Q32853). Libraries were sequenced using Illumina Novaseq instrument.
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina NovaSeq 6000
 
Data processing Sequencing reads mapping, quality filtering and the summary of DNA methylation level for each cytosine was performed as previously described in Luo et al., 2017 with minor modifications. Non-clonal mapped reads were filtered for MAPQ > 10 using samtools view -bq10 option.
Supplementary_files_format_and_content: tab delimited text files of methylcytosine calls; columns in allc files are: column 1 - chromosome; column 2 - position; column 3 - strand; column 4 - class; column 5 - mC reads; column 6 - total reads; column 7 - methylated (Boolean value indicating the result of statistical test for methylated cytosines)
 
Submission date Mar 28, 2018
Last update date Sep 20, 2018
Contact name Joseph R Ecker
E-mail(s) ecker@salk.edu
Phone 8584534100
Organization name HHMI-Salk-Institute
Department Genomic Analysis Laboratory
Lab Ecker lab
Street address 10010 North Torrey Pines Road
City La Jolla
State/province CA
ZIP/Postal code 92037
Country USA
 
Platform ID GPL24247
Series (1)
GSE112471 Robust single-cell DNA methylome profiling with snmC-seq2
Relations
BioSample SAMN08811621
SRA SRX3859798

Supplementary file Size Download File type/resource
GSM3070829_allc_180118_CEMBA_mm_P56_P63_4B_CEMBA180104_4B_1_CEMBA180104_4B_2_A12_AD006_indexed.tsv.gz 186.3 Mb (ftp)(http) TSV
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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