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Sample GSM5088641

Query DataSets for GSM5088641

Status

Public on Oct 27, 2021

Title

day14 NK cells-rep4

Sample type

SRA

Source name

primary natural killer cells

Organism

Homo sapiens

Characteristics

tissue: blood
cell type: primary natural killer cells
culture day: day14

Growth protocol

Isolated PBMCs were seeded on γ-globulin (Greencross, Yongin, Korea) and anti-NKp46 (R&D systems, Minneapolis, MN, USA) coated flask at a density of 2x106 cells/ml and cultured in Alys505NK EX serum free medium (CSTI, Sendai, Japan) supplemented with 1000 IU/ml recombinant human IL-2 (Novartis, Basel, Switzerland), 50 ng/ml recombinant human IL-18 (R&D system, Minneapolis, MN, USA) and 5% heat-inactivated autologous plasma. Fresh culture medium was added every 1 to 3 days depending on the cell density (2x106 cells/ml). The cells were transferred to culture bag (NIPRO, OSAKA, Japan) at 6 days, and cultured in a 5% CO2 incubator at 37°C for 14 days.

Extracted molecule

total RNA

Extraction protocol

In order to construct cDNA libraries with the TruSeq Stranded mRNA LT Sample Prep Kit, total RNA was used. The protocol consisted of polyA-selected RNA extraction, RNA fragmentation, random hexamer primed reverse transcription and 100nt paired-end sequencing by Illumina NovaSeq 6000.
A library was independently prepared with 1ug of total RNA for each sample by Illumina TruSeq Stranded mRNA Sample Prep Kit (Illumina, Inc., San Diego, CA, USA, #RS-122-2101). The first step in the workflow involves purifying the poly‐A containing mRNA molecules using poly‐T‐attached magnetic beads. Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using SuperScript II reverse transcriptase (Invitrogen, #18064014) and random primers. This is followed by second strand cDNA synthesis using DNA Polymerase I, RNase H and dUTP. These cDNA fragments then go through an end repair process, the addition of a single ‘A’ base, and then ligation of the adapters. The products are then purified and enriched with PCR to create the final cDNA library.
The libraries were quantified using KAPA Library Quantificatoin kits for Illumina Sequecing platforms according to the qPCR Quantification Protocol Guide (KAPA BIOSYSTEMS, #KK4854) and qualified using the TapeStation D1000 ScreenTape (Agilent Technologies, # 5067-5582). Indexed libraries were then submitted to an Illumina NovaSeq (Illumina, Inc., San Diego, CA, USA), and the paired-end (2×100 bp) sequencing was performed by the Macrogen Incorporated.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

We preprocessed the raw reads from the sequencer to remove low quality and adapter sequence before analysis and aligned the processed reads to the Homo sapiens (hg19) using HISAT v2.1.0 (KIM et al, 2015).
And then, transcript assembly of known transcripts was processed by StringTie v1.3.4d (Pertea, Mihaela, et al., 2015, 2016).
Base on the result of that, expression abundance of transcript and gene were calculated as read count or FPKM value (Fragments Per Kilobase of exon per Million fragments mapped) per sample.
The expression profiles are used to do additional analysis such as DEG (Differentially Expressed Genes).
Genome_build: The reference genome sequence of Homo sapiens (hg19) and annotation data were downloaded from the NCBI.
Supplementary_files_format_and_content: The sheet contains FPKM values of genes for 6 samples, 3 from day 0 and 3 from day 14. The fold change was calculated between day 0 and day 14. The statistical significances were calculated using ANOVA.

Submission date

Feb 17, 2021

Last update date

Oct 27, 2021

Contact name

Daun Jung

E-mail(s)

jhd2800@gmail.com

Phone

+82)10-9498-2801

Organization name

CHA Bundang Medical Center