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Sample GSM7717593

Query DataSets for GSM7717593

Status

Public on Jul 01, 2024

Title

4T1, Day4, single cell 17

Sample type

SRA

Source name

4T1

Organism

Mus musculus

Characteristics

host: Female BALB/c mice
cell line: 4T1
cell type: metastatic tumor cells

Extracted molecule

polyA RNA

Extraction protocol

Reverse transcription and cDNA amplification were carried out using a modified Smart-seq2 protocol (Picelli et al., 2014). Single cells were sorted by FACS as described above into 96-well plates containing 0.1 μl of RNase inhibitor (Clontech, Cat# 2313A), 1.9 μl of 0.2% Triton X-100 (Sigma, Cat# T9284), 1 μl of 10 µM oligo-dT primer (5′-AAGCAGTGGTATCA ACGCAGAGTAC T30VN-3′) and 1 μl of dNTP mix (10 mM each; Fermentas, Cat# R0192) in each well. Plates were either stored at -80 °C or processed immediately. Reverse transcription, PCR preamplification, PCR purification were performed following Picelli’s protocol (Picelli et al., 2014). As mRNA quality control, 1 ul of each PCR product was used as template for qPCR detection of mCherry expression and samples with Ct value higher than 30 will be discard. PCR products were purified with 1 × Agencourt Ampure XP beads (Beckman Coulter, Cat# A63881) and the final product was reconstituted in 20 µl TE buffer. The concentration of cDNA was measured by Qubit™ 1× dsDNA High Sensitivity (HS) Assay Kit (Invitrogen, Cat# Q33230). Tagmentation and NGS library was carried out by using TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, Cat# TD503) following the manufacturer’s instructions. The purified library was qualify controlled by measuring the fragment size distribution with an Agilent HS DNA BioAnalyzer Chip and the concentration of library was measured with Qubit™ 1× dsDNA High Sensitivity (HS) Assay Kits following the manufacturer’s instructions. Single cell cDNA libraries were sequenced on an Illumina HiSeq X Ten platform to obtain paired-end 150-bp reads.
SMART-Seq2 (Picelli et al., 2014)

Library strategy

RNA-Seq

Library source

transcriptomic single cell

Library selection

cDNA

Instrument model

HiSeq X Ten

Description

4T1_Day4_sc17

Data processing

Raw reads were first analyzed for quality control using FastQC (v0.11.7) (Andrews, 2010). Adaptor sequences of raw reads were removed by using TrimGalore (v0.6.6) (https://github.com/FelixKrueger/TrimGalore). Trimmed reads were mapped to a concatenated mouse genome (mm10) with STAR (v2.6.0c) with default parameters to result SAM format files (Dobin et al., 2013). Aligned reads stored in SAM format were transformed to generate BAM files using SAMtools (v1.7) (Danecek et al., 2021). The gene-level read count matrix was summarized from BAM files using FeatureCounts program (v1.6.2) (Liao et al., 2014). Subsequent data analysis was carried out in R 3.6.2 and the Seurat package (v3.1.2) (Stuart et al., 2019).
The gene counts per cell, the percentage of mitochondrial transcripts, the percentage of ERCC RNA spike-in, the percentage of α and β-globin genes were computed using the functions of the Seurat package. Quality controls were performed following: a. Cells with luciferase or GFP sequence detected were kept for further analysis. b. Cells with transcripts counts lower than 5 × 105 or higher than 1 ×107 were discarded. c. Single cell libraries with number of detected gene lower than 5,000, mapping rate lower than 40% or the percentage of mitochondrial transcripts higher than 10% were discarded before further analysis. d. Libraries with the percentage of α and β-globin genes higher than 1% were also discarded. 424 single cells passed quality controls, and were used for further analysis. The gene expression level was normalized as log2(TPM/10 + 1), in which TPM is the abbreviation for transcripts per million, and adding one to avoid negative expression values.
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files

Submission date

Aug 18, 2023

Last update date

Jul 01, 2024

Contact name

Hanqiu Zheng

E-mail(s)

hanzheng@tsinghua.edu.cn

Organization name

Tsinghua university

Street address

Haidian District