GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM3827151

Query DataSets for GSM3827151

Status

Public on Mar 27, 2020

Title

LN_05

Sample type

SRA

Source name

Normal lymph node

Organism

Homo sapiens

Characteristics

patient id: P2005
tumor stage: I
tissue origin abbrevation: nLN
lung cancer subtype: lung adenocarcinoma

Growth protocol

Single-cell suspensions of the collected tissues were prepared by mechanical dissociation and enzymatic digestion within 16 hours after the surgery without any in vitro culture.

Extracted molecule

polyA RNA

Extraction protocol

Single cell isolation was differently performed following each sample conditions. (1) Tumour and distant normal lung tissue dissociation was performed using tumor dissociation kit (Miltenyi Biotech, Germany) following the manufacturer’s instruction. Briefly, tissue was cut into 2-4 mm pieces and transferred to C tube containing the enzyme mix (Enzyme H, R, and A in RPMI1640 media). GentleMACS programs; h_tumor_01, 02, and 02 were run with two 30 min incubation in the MACSmix tube rotator at 37 oC. (2) Normal lymph node tissue and biopsy samples of metastatic lymph node and lung tumour tissue were dissociated using Collagenase/Hyaluronidase and DNase I. The tissue was chopped into 2-4 mm pieces with a sterile scissor in a 35 mm dish and incubated in an enzyme solution (Collagenase/Hyaluronidase (STEMCELL Technologies, Vancouver, Canada) and DNase I (QIAGEN, Hilden, Germany) in RPMI1640 media) at 37 oC for 1 hour. Tissue pieces are re-mixed by gentle pipetting at an interval of 20 min during incubation. (3) Metastatic brain tissue was chopped into 2-4 mm pieces with a sterile scissor in a 100 mm dish and incubated in an enzyme solution (Collagenase (Gibco, Waltham, MA, USA), DNase1 (Roche, Basel, Switzerland), and Dispase1 (Gibco, Waltham, MA, USA) in DMEM media) at 37 oC within 1 hour. Tissue pieces are re-mixed by gentle pipetting at an interval of 15 min during incubation. (4) Pleural fluids were transferred to a 50 ml tube and spun down at 300g. The cell suspension was transferred into a new 50 ml (or 15 ml; for biopsy samples) tube through 70um strainer. The volume in the tube was readjusted to 50 ml (or 15ml) with RPMI1640 media and spun down to remove enzymes. The supernatant was aspirated out and the cell pellet was resuspended in 4 ml of RPMI1640 media and dead cells were removed by Ficoll-Paque PLUS (GE Healthcare, Chicago, IL, USA) separation.
Each cell suspension was subjected to 3’ single-cell RNA sequencing using the GemCode system (10x genomics, Pleasanton, CA, USA) with cell recovery target at 5000, following the manufacturer’s instructions. Libraries were sequenced on an Illumina HiSeq2500.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

Single-cell RNA-seq
Lung_Cancer_raw_UMI_matrix.txt
Lung_Cancer_normalized_log2TPM_matrix.txt

Data processing

Fastq files were quantified to unique molecular identifiers (UMI) count values with Cell Ranger toolkit (version 2.1.0) with default parameters.
For each cell, we applied the three quality measures of mitochondrial genes (≤20%), and UMI and gene count (range from 100 to 150,000 and range from 200 to 10,000, respectively), which was applied to the raw gene-cell-barcode matrix.
UMI count for a gene in each cell was log-normalized to TPM-like values and then finally used in the scale of log2 (TPM+1), as previously described.
Genome_build: GRCh38
Supplementary_files_format_and_content: tab-delimited text files representing: UMI counts (Lung_Cancer_raw_UMI_matrix.txt); normalized data as described above (Lung_Cancer_normalized_log2TPM_matrix.txt)

Submission date

May 29, 2019

Last update date

Aug 18, 2021

Contact name

Hae-Ock Lee

E-mail(s)

haeocklee@catholic.ac.kr

Phone

82222588155

Organization name

College of Medicine, The Catholic University of Korea