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Sample GSM3825595

Query DataSets for GSM3825595

Status

Public on Oct 15, 2019

Title

Control2

Sample type

SRA

Source name

MNC

Organism

Homo sapiens

Characteristics

cell type: peripheral blood mononucleated cells
disease: acute leukemia
agvhd status: without

Extracted molecule

total RNA

Extraction protocol

Mononucleated cells was isolated from the peripheral blood. Total RNA of each sample was extracted using TRIzol Reagent (Invitrogen)/RNeasy Mini Kit (Qiagen)/other kits. Total RNA of each sample was quantified and qualified by Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), NanoDrop (Thermo Fisher Scientific Inc.) and 1% agrose gel. 1 μg total RNA with RIN value above 7 was used for following library preparation. Next generation sequencing library preparations were constructed according to the manufacturer’s protocol (NEBNext® Ultra™ RNA Library Prep Kit for Illumina®). Whole transcriptome sequencing libraries (3 pairs of aGvHD and controls, 6 libraries in total) were prepared following the manufacturer’s instructions for the Whole Transcriptome Sample Prep Kit (Illumina) by GENEWIZ Company.
RNA libraries were prepared following the manufacturer’s instructions for the Whole Transcriptome Sample Prep Kit (Illumina) by GENEWIZ Company. The poly(A) mRNA isolation was performed using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB) or Ribo-Zero™ rRNA removal Kit (illumina). The mRNA fragmentation and priming was performed using NEBNext First Strand Synthesis Reaction Buffer and NEBNext Random Primers. First strand cDNA was synthesized using ProtoScript II Reverse Transcriptase and the second-strand cDNA was synthesized using Second Strand Synthesis Enzyme Mix. The purified double-stranded cDNA（by AxyPrep Mag PCR Clean-up (Axygen)was then treated with End Prep Enzyme Mix to repair both ends and add a dA-tailing in one reaction, followed by a T-A ligation to add adaptors to both ends. Size selection of Adaptor-ligated DNA was then performed using AxyPrep Mag PCR Clean-up (Axygen), and fragments of ~360 bp (with the approximate insert size of 300 bp) were recovered. Each sample was then amplified by PCR for 11 cycles using P5 and P7 primers, with both primers carrying sequences which can anneal with flow cell to perform bridge PCR and P7 primer carrying a six-base index allowing for multiplexing. The PCR products were cleaned up using AxyPrep Mag PCR Clean-up (Axygen), validated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), and quantified by Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

HiSeq X Ten

Data processing

Image analysis and base calling were conducted by the HiSeq Control Software (HCS) + OLB + GAPipeline-1.6 (Illumina) on the HiSeq instrument.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, and all sequencing reads were mapped to Human genome using tophat.
The mapped results were processed using cufflinks, producing Fragments per Kilobase of transcript per Million mapped reads data matrix, and with htseq-count(http://www-huber.embl.de/users/anders/HTSeq/doc/count.html), producing the raw hit counts data matrix. The raw hit counts gene matrixes were transformed using DESeq variance stabilization algorithm and normalized using quantile normalization function built into the Bioconductor Limma package. The normalized data matrix was then analyzed using Limma and SAMR algorithms.
Genome_build: hg19
Supplementary_files_format_and_content: text file with RPKM

Submission date

May 29, 2019

Last update date

Oct 15, 2019

Contact name

Aijie Huang

E-mail(s)

huangaijie@mail.ustc.edu.cn

Organization name

Changhai Hospital

Street address

168, Changhai road