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Sample GSM4981254

Query DataSets for GSM4981254

Status

Public on Dec 19, 2021

Title

sncRNA organ atlas, Mus musculus, Brain_11

Sample type

SRA

Source name

Brain_11

Organism

Mus musculus

Characteristics

strain: C57/BL6
age (months postnatal): 21
developmental stage: postnatal
Sex: male
tissue: Brain

Growth protocol

The bodies were stored at 4°C upon arrival at the anatomical institute and tissue samples were collected between 8 and 48 hours post-mortem. To increase the resolution for selected organs, e.g. brain or intestine, further dissection of these organs was performed to include several subareas. After collection, samples were immediately stored in RNALater (Sigma-Aldrich) or frozen at -80 °C to prevent further degradation. Mice tissues were prepared as described for GSE132042.

Extracted molecule

total RNA

Extraction protocol

Total RNA including smallRNAs was isolated using the miRNeasy Mini Kit (Qiagen) according to the manufacturer’s recommendations. In brief, tissues were lysed and homogenized in 700 µl QIAzol Lysis Reagent using 5 mm stainless steel beads and the TissueLyser LT (Qiagen) for 5 min at 50 Hz. After 5 min incubation, samples were mixed with 140 µl chloroform and phase separation was performed at 12000 x g and 4 °C for 15 min. The RNA in the aqueous phase was precipitated by adding 1.5 Volume of 100 % ethanol and purified using the RNeasy columns provided with the kit, either manually or semi-automated using the Qiacube instrument with the respective protocols provided for the miRNeasy Mini kit.
We performed library preparation for sequencing on the BGISEQ-500RS following the instructions of the MGIEasy Small RNA Library Prep Kit (MGI Technologies). To limit the bias the manual library preparation performed previously was replaced by a semi-automated workflow on the MGI-SP100. In general, 100 ng total RNA was used as input material for the library preparation. The library construction includes of 3’ adapter ligation, 3’ adapter digestion, 5’ adapter ligation followed by reverse transcription (RT) of RNA into cDNA using RT primers which contained specific barcodes. Next, cDNA was amplified by PCR in 22 cycles. The PCR products were separated using 6 % TBE gels and bands of the size around 110 bp were cut from the gel. After purification, the DNA yield was measured with the Qubit® 1xdsDNA HS Assay Kit (Invitrogen). A total of 16 different barcoded samples were pooled together in equal amounts and circularized to generate a single-stranded DNA library. The BGISEQ-500RS uses DNA nanoballs (DNBs) for sequencing, which were generated by rolling circle amplification (RCA). DNBs were then loaded into the patterned nanoarray using the BGIDL-50 DNB loader. Finally, single-end sequencing was performed on the BGISEQ-500RS using the High-throughput Sequencing Set (SE50) (Small RNA) according to the manufacturer’s instructions. The mouse samples have been processed with an advanced pipeline using the higher-throughput SP-960 sample prep system and MGISEQ-400 sequencing system. In the latter case we made further use of the CoolMPS sequencing chemistry.

Library strategy

ncRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

DNBSEQ-G400

Data processing

We performed the primary data analysis using a stand-alone version of our web-based tool miRMaster, version 1.1. We carried out all analyses using the standard parameter settings as in the online version, allowing one mismatch in the quantification of ncRNAs. The following ncRNA databases are included in our analysis: miRBase version 22.1, Ensembl ncRNA version 100, RNACentral version 15 and GtRNAdb version 18.1.
Adapters at the 3′ end were trimmed, while allowing an error of maximum one base and requiring a minimum overlap with the read of 10 bases. Reads were quality trimmed when the average quality dropped below 20 in a window of four consecutive bases to ensure a high quality of reads used for the downstream processing. All reads shorter than 17 bases after trimming were discarded from all further analyses.
MiRNAs were quantified with up to one mismatch and a variability of two bases allowed at the 5′ end and five bases at the 3′ end.
Genome_build: hg38, mm10
Supplementary_files_format_and_content: sncRNA_counts_human.txt: tab-delimited text file containing the RPMM normalized counts of all quantified sncRNAs in the human samples.
Supplementary_files_format_and_content: sncRNA_counts_mouse.txt: tab-delimited text file containing the RPMM normalized counts of all quantified sncRNAs in the mouse samples.

Submission date

Dec 18, 2020

Last update date

Dec 19, 2021

Contact name

Tobias Fehlmann

Organization name

Saarland University

Street address

Campus E2 1

City

Saarbrücken