GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM8481115

Query DataSets for GSM8481115

Status

Public on Aug 31, 2024

Title

CFS, Diestrus3

Sample type

SRA

Source name

cell free Saliva

Organism

Bubalus bubalis

Characteristics

tissue: cell free Saliva
animal id: 3
genotype: Wildtype
phase: Diestrus

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted from CFS using TRIzol LS (Life Technologies, USA) as per the manufacturer's protocol. In brief, the mix of CFS and TRIzol LS was thawed on ice and kept at room temperature for 5 min. 200ul chloroform was added per 1ml of Trizol LS used and the mixture was vortexed for 20 secs followed by incubation at room temperature for 5 minute and then centrifuged at 12000g for 15 minutes. Aqueous layer containing RNA was transferred to fresh Eppendorf tube and added 1ul of linear polyacrylamide, 0.1X of 3M NaOAc and 2.5X of absolute ethanol and kept at -80 O/N and finally centrifuged at 14000g for 30 minutes to get RNA pellet. Further processing of total RNA was performed as per manufacturer instructions and finally the extracted total RNA was DNaseI digested, purified using Zymo Columns, and final RNA elution was done in 10 μL of nuclease-free water and stored at −80°C. Total RNA was sent to Genotypic Pvt Ltd, Bangalore, India for custom NGS Library preparation and subsequent generation of miRNA Seq data. RNA purity and concentrations were determined using Nanodrop spectrophotometer, Qubit and Bioanalyzer available at Genotypic Pvt Ltd, Bangalore, India.
Small RNA sequencing libraries were prepared with QIAseq® miRNA Library Kit (Cat: 331502) protocol (Qiagen, Maryland, USA). In brief, 5 ul of total RNA was used as starting material. 3’ adapters were ligated to the specific 3’OH group of miRNAs followed by ligation of 5’ adapter. Adapter ligated fragments were reverse transcribed with Unique Molecular Index (UMI) assignment by priming with reverse transcription primers. cDNA thus formed was enriched and barcoded by PCR amplification (22cycles). The 5’ and 3’ adapters sequences used in the protocol are 5’-GTTCAGAGTTCTACAGTCCGACGATC-3’ and 5’-AACTGTAGGCACCATCAAT-3’ respectively. Universal Illumina adapter sequence used in the protocol is 5’-AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3’. Qubit fluorometer (Thermo Fisher Scientific, MA, USA) was used to quantify sequencing library. Agilent 2100 Bioanalyzer was used to analyze the fragment size distribution. The raw miRNA Seq data of Bubalus Bubalis cell free saliva samples were deposited in National Center for Biotechnology Information (NCBI) GEO database with accession no XXXXXXX. All libraries were sequenced through Illumina Next Seq with 75 bp reads in order to generate greater than 10 million SE reads per sample as per manufacturer's instructions.

Library strategy

miRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina NovaSeq 6000

Description

SO_10224_779617121

Data processing

miRNA Seq data was analyzed through various open-source softwares as described previously [34]. Reference mature miRNA file was downloaded from mirBase whereas genome fasta and GFF files were downloaded from NCBI. Reference indexes were created using bowtie build command. FastQC tool was used to assess the overall quality of each sample. UMIs were extracted from the reads and adaptors were removed using UMI_tools as mentioned previously [34]. UMI extracted and adaptor removed reads were filtered to retain reads for downstream miRNA analysis with read length >15 bp and <30 bp using Cutadapt tool. Filtered reads were aligned initially to known bta mature miRNA sequences from miRBase (v22) using Bowtie (with parameters -n 1 -l 19 --norc --best --strata -a). Unaligned reads from the previous step were aligned to the Bos Taurus reference genome GCF_002263795.3 using Bowtie (with parameters -n 1 -l 19 --norc --best --strata -m 1). Samtools view option were used to sort and convert the SAM file into BAM file. BAM files were deduplicated using the UMI_tools dedup option –method=unique. Samtools idxstats were used to count each miRNA in every sample aligned to miRNAs whereas featurecounts tools were used to count reads aligned to miRNA coordinates in the reference genome using miRNA subset of GFF annotation file. Both mature miRNA-based counts and genome-based miRNA counts were combined into one file using R. DESeq2 was used to identify differentially expressed miRNAs and pheatmap function was used to create heatmap of log2 normalized counts.
Assembly: Bos Taurus reference genome GCF_002263795.3
Supplementary files format and content: Normalized_Counts in csv format

Submission date

Aug 25, 2024

Last update date

Aug 31, 2024

Contact name

Suneel Kumar Onteru

E-mail(s)

[email protected]

Organization name

NDRI

Department

Animal Biochemistry