Stranded Illumina libraries for each tissue were prepared from total RNA using the NEB Ultra Directional RNA library prep kit with poly(A) selection. Sequencing (paired-end 100 bp) was performed on the Illumina HiSeq-2000 platform at the UC Denver Genomics core.
Neb ultra directional rna library prep kit
Manufactured by New England Biolabs
The NEB Ultra Directional RNA library prep kit is a laboratory tool used to prepare directional RNA libraries for sequencing. The kit provides the necessary reagents and protocols to convert RNA samples into cDNA libraries suitable for next-generation sequencing platforms.
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3 protocols using neb ultra directional rna library prep kit
1
Illumina RNA-Seq Library Preparation
2
RNA-seq of antibiotic-treated samples
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Total RNA was isolated from cultures after antibiotics treatment using Tri-Reagent (Sigma). After additional DNAse digestion, RNA samples were depleted for eukaryotic and bacterial rRNAs by subsequent usage of the Yeast Ribo-Kit and the gram-negative bacterial Ribo-Zero Kits (Illumina). After additional depletion of poly(A) RNAs, directional RNA libraries were generated from the left-over RNA using the NEB ultra directional RNA library prep Kit (NEB). Libraries were sequenced on a Illumina HiSeq2500 Platform and trimmed as described above. RNA reads have been deposited under ENA accession number PRJEB36201.
3
Transcriptome Analysis of Hair Follicle Samples
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To extract the total RNA from the hair follicle samples, TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was utilized according to the manufacturer's instructions. Subsequently, RNA integrity, purity, and concentration were estimated using 1% agarose gels and spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA). First, an EpicentreRibo-Zero™ rRNA Depletion Kit (Epicentre, WI, USA) was employed to remove ribosomal RNA. Subsequently, RNA sequencing (RNAseq) libraries were generated using the NEB Ultra™ Directional RNA Library Prep kit (New England BioLabs, Inc.). Moreover, the libraries were sequenced on Hiseq X Ten (Illumina, San Diego, CA, USA). The Fast-QC (http:// www. bioin forma tics. babra ham. ac. uk/ proje cts/ fastqc/) software was employed to evaluate the overall quality of sequencing data. The HISAT2 software was used to compare clean reads with the reference genome GRCg6a. The DEGs were screened by utilizing DESeq2 on the basis of |log2(Fold change)|> 1 and P value < 0.05. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were utilized to analyze the function and pathways of DEGs.
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