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Sample GSM6970155

Query DataSets for GSM6970155

Status

Public on Jun 21, 2023

Title

Control_NK_rep2 RNA-seq

Sample type

SRA

Source name

Organism

Homo sapiens

Characteristics

cell line: YT
cell type: natural-killer-like (NK-like) lymphoid cells
genotype: eGFP control expressing
treatment: lentiviral vector infection

Extracted molecule

total RNA

Extraction protocol

Total RNA samples isolated from CTRL/hnCD16/FR1/FR2 YT cells were treated with DNase using a column assay. High-quality RNA (280/260 and 230/260 both over 1.7, RIN/RQN>9) was subjected to ribosomal RNA depletion followed by library construction.
Sample testing: According to the sample and product requirements, select the corresponding testing method for quality inspection. mRNA isolation: A certain amount of RNA sample was taken, denatured at a suitable temperature to open its secondary structure, and mRNA was enriched using oligo(dT) magnetic beads. mRNA interruption: Add an interruption reagent to the mRNA obtained in the previous step, react at an appropriate temperature for a certain period of time, and fragment the mRNA. cDNA synthesis: Prepare the first-strand synthesis reaction system and set the reaction program to synthesize the first-strand cDNA; prepare the second-strand synthesis reaction system and set the reaction program to synthesize the second-strand cDNA. Prepare the reaction system for end repair, add "A" and adapter ligation, and set the reaction program to repair the double-stranded cDNA end, and add A base at the 3' end; prepare the adapter ligation reaction system, and set the reaction program to make the adapter Ligated with cDNA. PCR Prepare a PCR reaction system and set up a reaction program to amplify the product. Library detection: Select the corresponding detection method according to the product requirements to check the quality of the library. Circularization: After denaturing the PCR product into a single strand, prepare a circularization reaction system and set up a reaction program to obtain a single-stranded circular product and digest the uncircularized linear DNA molecules. On-machine sequencing: The single-stranded circular DNA molecule is replicated by rolling circle to form a DNA nanoball (DNB) containing multiple copies. The obtained DNBs are added to the mesh holes on the chip using high-density DNA nanochip technology, and sequenced by combined probe-anchored aggregation technology (cPAS).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

DNBSEQ-T7

Description

CTRL_2

Data processing

Data filtering The raw data obtained by sequencing was filtered using SOAPnuke (v1.5.6)
Differential gene analysis Use HISAT2 (v2.1.0) software to compare the clean data to the reference genome.
Use Bowtie2 (v2.3.4.3) to align the clena data to the reference gene set.
RSEM (v1.3.1) software was used to quantify gene expression, and pheatmap (v1.0.8) was used to draw a cluster heat map of gene expression in different samples.
DESeq2 (v1.4.5) was used for differential gene detection, and the condition was Q value≤0.05 or FDR≤0.001.
Assembly: GCF_000001405.39_GRCh38.p13

Submission date

Jan 24, 2023

Last update date

Jun 21, 2023

Contact name

fanyi meng

E-mail(s)

[email protected]

Organization name

peking university

Street address

haidian distinct