RNA sequencing of the nephron transcriptome: a technical note

Kidney Research and Clinical Practice
Jae Wook Lee

Abstract

To understand the functions of the kidney, the transcriptome of each part of the nephron needs to be profiled using a highly sensitive and unbiased tool. RNA sequencing (RNA-seq) has revolutionized transcriptomic research, enabling researchers to define transcription activity and functions of genomic elements with unprecedented sensitivity and precision. Recently, RNA-seq for polyadenylated messenger RNAs [poly(A)'-mRNAs] and classical microdissection were successfully combined to investigate the transcriptome of glomeruli and 14 different renal tubule segments. A rat kidney is perfused with and incubated in collagenase solution, and the digested kidney was manually dissected under a stereomicroscope. Individual glomeruli and renal tubule segments are identified by their anatomical and morphological characteristics and collected in phosphate-buffered saline. Poly(A)'-tailed mRNAs are released from cell lysate, captured by oligo-dT primers, and made into complementary DNAs (cDNAs) using a highly sensitive reverse transcription method. These cDNAs are sheared by sonication and prepared into adapter-ligated cDNA libraries for Illumina sequencing. Nucleotide sequences reported from the sequencing reaction are mapped to the rat refe...Continue Reading

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Citations

Aug 19, 2016·Clinical Immunology : the Official Journal of the Clinical Immunology Society·Celine C BerthierAnne Davidson

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Methods Mentioned

BETA
RNA-seq
dissecting
dissector
PCR
Illumina sequencing

Software Mentioned

TopHat2 Aligner ( BWA
count
DESeq2
FastQC
BEDTools
Illumina pipeline
FASTA
edgeR
DESeq
Bowtie

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