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Sample GSM4002796 Query DataSets for GSM4002796
Status Public on Aug 01, 2020
Title 0.5ng RNA input rep1
Sample type SRA
Source name 0.5ng RNA
Organism Mus musculus
Characteristics strain: C57
age: 6-18week
cell line: raw 264.7
Extracted molecule total RNA
Extraction protocol RNA was isolated from different number of cell samples by using the Direct-zolTM RNA MiniPrep Kit
The qualified RNA samples were added with 1 µl of SuperScript III reverse transcriptase, 4 µl of Superscript III first strand synthesis buffer , 1 µl of dNTP mixture, 1 µl of Oligo 30 (dT) primer , 1 µl of RNase inhibitor and 1 µl of DTT (0.1M) to construct 20 µl reaction system for RNA reverse transcription by heating the mixtures for 50 ℃ for 90 min, 70 ℃ for 15min, and a final hold at 4 ℃. For second strand cDNA synthesis, the 20 µl products of reverse transcription were directly mixed into reaction buffer on ice to obtain dsDNA samples with the help of DNA polymerase I and T4 DNA ligase, following the manufacturer’s recommendations . dsDNA products obtained from the synthesis were purified with 144 µl (1.8 ×) of Agencourt AMPure XP Beads through magnetic separation. The purified dsDNA samples were then dissolved into nuclease-free water and diluted to 1 ng/µl following Qubit 2.0 measurement . 1 ng of dsDNA, and 4 µl of 5 × TTBL and 5 µl of TTE Mix V1 were used to in the Tn5 tagmentation reaction system. Reaction was performed for 10 mintues at 55℃ and quenched with 5 µl of pre-mixed 5 × TS to avoid excessive DNA fragmentation. Samples were then barcoded following the protocol of TruePrep DNA Library Prep Kit for Illumina with P5/P7 adapter primers , and amplicon purified via the VAHTSTM DNA clean beads kit.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model HiSeq X Ten
Data processing Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mm8 whole genome using Hisat2 v2.1.0 with parameters -p 16
Use featurecount to calculate the count to each gene
Genome_build: mm20
Supplementary_files_format_and_content: tab-delimited text files include row count values for each Sample
Submission date Aug 02, 2019
Last update date Aug 01, 2020
Contact name peng zhou
Organization name First Affiliated Hospital of Army Medical University
Street address 重庆市大渡口区天安数码城5栋10层
City chongqing
ZIP/Postal code 400000
Country China
Platform ID GPL21273
Series (1)
GSE135290 Streamlined low-input transcriptomics through EASY-RNAseq
BioSample SAMN11646869
SRA SRX5832230

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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