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| Status |
Public on Sep 20, 2018 |
| Title |
171219_CEMBA_mm_P56_P63_4B_CEMBA171213_4B_1_CEMBA171213_4B_2_A2_AD004_indexed |
| Sample type |
SRA |
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| Source name |
Mouse primary motor cortex
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| Organism |
Mus musculus |
| Characteristics |
Sex: Male
age: P56-P63
genome build: mm10
library strategy: snmC-seq2
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| 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.
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| 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.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| 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)
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| Submission date |
Mar 28, 2018 |
| Last update date |
Sep 20, 2018 |
| Contact name |
Joseph R Ecker |
| E-mail(s) |
ecker@salk.edu
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| Phone |
8584534100
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| Organization name |
HHMI-Salk-Institute
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| Department |
Genomic Analysis Laboratory
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| Lab |
Ecker lab
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| Street address |
10010 North Torrey Pines Road
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE112471 |
Robust single-cell DNA methylome profiling with snmC-seq2 |
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| Relations |
| BioSample |
SAMN08811458 |
| SRA |
SRX3859780 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3070811_allc_171219_CEMBA_mm_P56_P63_4B_CEMBA171213_4B_1_CEMBA171213_4B_2_A2_AD004_indexed.tsv.gz |
140.7 Mb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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