"mitoRCA-Seq_CK1, comparing libraries derived from 5ng": Sample Liver... - SRA

SRX1001754: "mitoRCA-Seq_CK1, comparing libraries derived from 5ng": Sample LiverMut_5ng
1 ILLUMINA (Illumina MiSeq) run: 4.2M spots, 424M bases, 190.9Mb downloads

Design: We constructed mitoRCA-seq library starting from total DNA extracted from Brain and live tissues were taken from wild-type and Polg mutant mice (C57BL/6) of 6 weeks old or the control plasmid. For constructing mitoRCA-seq library, 100 ng of total DNA was used as template for RCA amplification. Briefly, a 50 μl reaction mix containing 100 ng total DNA, 1x Phi29 buffer (NEB), 0.2 μg/ml BSA, 1 mM dNTP (NEB) and 25 μM specific oligos (20 primer pairs for the pTEsindbisGFP plasmid, 19 primer pairs for mouse mtDNA and 13 primer pairs for Drosophila mtDNA) was denatured for 3 min at 95°C. After cooling down at room temperature for 10 minutes, 1 μl of Phi29 DNA polymerase (10 unit/μl, NEB) was added to the reaction mix, which was then incubated at 37°C for 16 hours followed by 65°C for 10 minutes to heat inactivate the enzyme. For the titration test in Supplementary Figure 1, the input DNA amounts were titrated from 100 ng to 0.1 pg. The REPLI-g Mitochondrial DNA kit (Qiagen), which is based on RCA amplification, can also be used to selectively enrich human and mouse mtDNAs from total genomic DNA. Then, different restriction enzymes were selected to digest the RCA products according to the species’ mtDNA reference sequence to generate two distinguishable bands in 0.5% agarose gel. For mouse, EcoRV (NEB) was used to digest RCA products into two long fragments (9.5kb and 6.8kb). For fruit fly, NdeI (NEB) and EcoRV (NEB) were used to digest RCA products into two long fragments (10kb and 9.8kb). For plasmid pTEsindbisGFP, EcoRV (NEB) was used to digest RCA products into two long fragments (10.4kb and 4.1kb). All digestion reactions were carried out according to the manufacturer’s protocol. Size selection of mitochondrial specific DNA fragments was carried out with 0.5% agarose gel. Zymoclean large fragment gel purification kit (ZYMO research) was used to purify the excised bands. The resulting DNA fragments were then sheared by Covaris S2 instrument into small fragments with a size peaked at 300 bp. End-IT kit from Epicentre was applied to perform end repairing, followed by size selection for 200-400bp in a 2% agarose gel. A-tailing of the blunt-ended DNA was accomplished with Klenow exo- DNA polymerase (Epicentre). Illumina index adaptors were then ligated, followed by 8-12 cycle PCR with Phusion High Fidelity DNA polymerase (NEB). Size selection (300-500bp) of the final PCR products was performed in a 2% agarose gel to prepare the mitoRCA-seq libraries, which were subjected to deep sequencing by Illumina HiSeq2000 or MiSeq instrument. For PCR-free libraries, we followed the library construction protocol of NuGEN Encore Rapid Library Systems after the fragmentation step.

Submitted by: School of Life sciences, Fudan University, Shangha

Study: Mus musculus Raw sequence reads

PRJNA280391 • SRP056905 • All experiments • All runs

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DNA-seq was performed on brain and liver tissues taken from wild-type and Polg mutant mice of 6 weeks old, in order to find out the mitochondrial mutation difference and figure out the special feature of mitochondiral mutations in Polg mutant comparing to wild-type ones.

Sample: LiverMut_5ng

SAMN03463174 • SRS915683 • All experiments • All runs

Organism: Mus musculus

Library:

Name: LiverMut_5ng

Instrument: Illumina MiSeq

Strategy: WGS

Source: GENOMIC

Selection: size fractionation

Layout: PAIRED

Spot descriptor:

1 forward51 reverse

Runs: 1 run, 4.2M spots, 424M bases, 190.9Mb

Run	# of Spots	# of Bases	Size	Published
SRR1970527	4,240,309	424M	190.9Mb	2015-04-22

ID:: 1454923

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