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Sample GSM4002784

Query DataSets for GSM4002784

Status

Public on Aug 01, 2020

Title

100cells input rep3

Sample type

SRA

Source name

100 cells

Organism

Mus musculus

Characteristics

strain: C57
age: 6-18week
cell type: spleen T cell

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

E-mail(s)

zhoupeng@tcrcure.com

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