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Sample GSM7316678

Query DataSets for GSM7316678

Status

Public on May 10, 2024

Title

PYMT cells, BMP4-TGFB2_2

Sample type

SRA

Source name

PYMT

Organism

Mus musculus

Characteristics

cell line: PYMT
treatment: BMP4-TGFB2

Growth protocol

PYMT cells were treated with 5ng/mL of TGFΒ2, BMP4, or both for 3 days, then media was changed, and cells were collected 2 days later (day3+2) after matrigel depolymerization (Corning, CLS354253) following the manufacturer’s instructions. 3 biological replicates were performed, and then RNA extraction, sample QC, library preparations, and sequencing reactions were conducted at GENEWIZ

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted using the Qiagen RNeasy Plus Universal kit following the manufacturer’s instructions (Qiagen). Extracted RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies), and RNA integrity was checked using Agilent TapeStation 4200 (Agilent Technologies).
RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina following the manufacturer’s instructions (NEB). Briefly, mRNAs were first enriched with Oligo(dT) beads. Enriched mRNAs were fragmented for 15 minutes at 94 °C. First- strand and second-strand cDNAs were subsequently synthesized. cDNA fragments were end-repaired and adenylated at 3’ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by limited-cycle PCR.
The sequencing libraries were validated on the Agilent TapeStation (Agilent Technologies), and quantified by using Qubit 2.0 Fluorometer (Invitrogen) as well as by quantitative PCR (KAPA Biosystems). The sequencing libraries were clustered on a flowcell. After clustering, the flowcell was loaded on the Illumina HiSeq instrument (4000 or equivalent) according to the manufacturer’s instructions. The samples were sequenced using a 2x150bp Paired-End (PE) configuration. Image analysis and base calling were conducted by Illumina Control Software.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Data processing

Raw sequence data (.bcl files) generated from the Illumina instrument was converted into fastq files and de- multiplexed using Illumina's bcl2fastq 2.17 software. One mismatch was allowed for index sequence identification.
The mouse genome reference used was GRCm38.p6 and GENCODE release M25 was used as the transcriptome reference. The alignment is performed by using STAR aligner (v2.7.5b)41. Gene level read counts are obtained by using Salmon (v1.2.1) for all libraries. All samples have passed the quality control requirements with > 85% of reads uniquely mapping (>25M uniquely mapped reads for each library) using STAR aligner.
Assembly: The mouse genome reference used was GRCm38.p6 and GENCODE release M25 was used as the transcriptome reference.
Supplementary files format and content: Tab-separated values files and matrix files

Submission date

May 10, 2023

Last update date

May 10, 2024

Contact name

Julio A Aguirre-Ghiso

Organization name

Albert Einstein College of Medicine

Department

Cell Biology

Street address

1301 Morris Park Ave