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Sample GSM5673277

Query DataSets for GSM5673277

Status

Public on Mar 05, 2024

Title

mTGN_Ctrl_2

Sample type

SRA

Source name

mTGN_Ctrl

Organism

Mus musculus

Characteristics

cell type: mouse trigeminal ganglion neurons
treatment: untreated

Treatment protocol

phKCs were treated with IL-27(100 ng/ml) for 6h or BST2(250 ng/ml) for 24h; mTGNs were treated with IL-27(100 ng/ml) or BST2(500 ng/ml) for 6h

Growth protocol

phKCs were seeded into 24 well plates and cultured in KBM-Gold medium with KBM Gold SingleQuot KC supplement (Lonza, Basel, Switzerland). Cells were maintained in the above medium for 3 days before use. Mouse trigeminal neurons (mTGNs) were isolated from postnatal d5 C57BL/6 mice after the mice after being deeply anesthetized by i.p. injection of a mixture of 0.2% xylazine, 1% ketamine, and 0.9% NaCl. The harvested ganglia were incubated with Hank’s balanced salt solution (HBSS) using collagenase I (10 mg/mL, Sigma) and dispase II (5 mg/mL, Sigma) for 30 minutes. Digests were washed with DMEM and the cell suspension was filtered through a 100-μm cell strainer and cultured in 24-well plates pre-coated with poly-L-lysine (Sigma) and laminin (Sigma) in medium consisting DMEM supplemented with 5% (v/v) FBS (Sigma), 100 U/ml penicillin, 100 μg/ml streptomycin in the presence of cytosine β-d-arabinofuranoside (Sigma) 10 µM, 1xB27, and nerve growth factor 50 ng/ml (Sigma) for 7 days

Extracted molecule

total RNA

Extraction protocol

Remove the upper medium and wash it twice with cold PBS, frozen on ice, and RNA was harvested using Trizol reagent.The mix was centrifuged for 5 minutes at 12,000×g at 4°C, then the supernatant was transferred into a new EP tube with 0.3 mL chloroform/isoamyl alcohol (24:1). The mix was shacked vigorously for 15s, and then centrifuged at 12,000×g for 10 minutes at 4°C. After centrifugation, the upper aqueous phase where RNA remained was transferred into a new tube with equal volume of supernatant of isopropyl alcohol, then centrifuged at 13,600 rpm for 20 minutes at 4°C. After deserting the supernatant, the RNA pellet was washed twice with 1 mL 75% ethanol, then the mix was centrifuged at 13,600 rpm for 3 minutes at 4°C to collect residual ethanol, followed by the pellet air dry for 5-10 minutes in the biosafety cabinet. Finally, 25µL~100µL of DEPC-treated water was added to dissolve the RNA. Subsequently, total RNA was qualified and quantified using a Nano Drop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA).
Oligo(dT)-attached magnetic beads were used to purified mRNA. Purified mRNA was fragmented into small pieces with fragment buffer at appropriate temperature. Then First-strand cDNA was generated using random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis. afterwards, A-Tailing Mix and RNA Index Adapters were added by incubating to end repair. The cDNA fragments obtained from previous step were amplified by PCR, and products were purified by Ampure XP Beads, then dissolved in EB solution. The product was validated on the Agilent Technologies 2100 bioanalyzer for quality control. The double stranded PCR products from previous step were heated denatured and circularized by the splint oligo sequence to get the final library. The single strand circle DNA (ssCir DNA) was formatted as the final library. The final library was amplified with phi29 to make DNA nanoball (DNB) which had more than 300 copies of one molecular, DNBs were loaded into the patterned nanoarray and single end 50 bases reads were generated on DNBSeq platform (BGI-Shenzhen, China)

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

MGISEQ-2000RS

Description

processed data file:
mTGNs_IL27_gene_expression.xls

Data processing

Base calling and the quality score of each base are calculated based on the phred-33 quality score standard by the sub-Pixel Registration algorithm according to the optical signal intensity of each lane
This project uses the filtering software SOAPnuke (Versionv1.5.2) developed by BGI independently for filtering. SOAPnuke Official Websitehttps://github.com/BGI-flexlab/SOAPnuke The specific steps are as follows: 1) Remove the reads containing the adaptor (adaptor pollution); 2) Remove reads whose N content is greater than 5%; 3) Remove low-quality reads (we define reads with bases with a quality score less than 10 as the proportion of total bases in the reads that are greater than 20% as low-quality reads).
Hierarchical Indexing for Spliced Alignment of Transcripts (HISTAT) is software for mapping RNA-seq reads. HISAT will use the global FM index to anchor the position of partial sequences in each read on the genome; then, use the partial genome indexes of these alignment positions to align the remaining sequences of each read to extend the alignment area. Software information:HISAT2 (Version：v2.0.4) ,Official Website：http://www.ccb.jhu.edu/software/hisat
Bowtie2 (v2.2.5) was used to compare clean reads to reference sequences for gene alignment with -q --phred64 --sensitive --dpad 0 --gbar 99999999 --mp 1,1 --np 1 --score-min L,0,-0.1 -p 16 -k 200. And then RSEM (v1.2.8) was used to calculate gene and transcript expression levels with -p 8 --forward-prob 0.5 --paired-end.
Genome_build: GCF_000001635.26_GRCm38.p6 for Mus musculus GCF_000001405.39_GRCh38.p13 for Homo sapiens
Supplementary_files_format_and_content: FPKM values, Reads Count for each Sample

Submission date

Nov 04, 2021

Last update date

Mar 05, 2024

Contact name

Yanqing Li

E-mail(s)

104752190132@henu.edu.cn

Phone

18237840178

Organization name

Henan University