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Sample GSM7026693

Query DataSets for GSM7026693

Status

Public on Mar 01, 2023

Title

Day 2 of hFOB differentiation replicate 1 [t2a]

Sample type

SRA

Source name

hFOB 1.19

Organism

Homo sapiens

Characteristics

cell line: hFOB 1.19
cell type: Human fetal osteoblasts

Growth protocol

hFOB 1.19 cells (American Type Culture Collection [ATCC], Manassas, VA, USA; #CRL-11372) were cultured at 34C and differentiated at 39.5C as recommended with the following modifications. Growth media: minimal essential media (MEM; Gibco, Grand Island, NY, USA; 10370-021) supplemented with 10% fetal bovine serum (FBS; Atlantic Biologicals, Morrisville, NC, USA; S12450), 1% Glutamax (Gibco; 35050-061), 1% Pen Strep (Gibco; 15140-122). Differentiation media: MEM alpha (Gibco; 12571-063) supplemented with 10% FBS, 1% Glutamax, 1% Pen Strep, 50 μg/μL Ascorbic Acid (Sigma-Aldrich, St. Louis, MO, USA; A4544-25G), 10mM beta-Glycerophosphate (Sigma-Aldrich; G9422-100G), 10nM Dexamethasone (Sigma-Aldrich; D4902-25MG). RNA was isolated from ~0.5 × 106 cells at days 0, 2, 4, and 10 of differentiation as recommended (RNAeasy Minikit; QIAGEN, Valencia, CA, USA; 74106). Mineralized nodule formation was measured by staining cultures with Alizarin Red (40mM, pH 5.6; Sigma-Aldrich; A5533-25G). Reported results were obtained from three biological replicate experiments for days 2,4 and 10, and two biological replicates for day 0 of differentiation

Extracted molecule

total RNA

Extraction protocol

RNA was extracted from 3 biological replicates of hFOB cells at days (0,2,4, and 10) using Qiagen RNeasy mini kit. Extracted RNA was used to create full length cDNA using the NEBNext® Single Cell/Low Input cDNA Synthesis & Amplification kit (New England Biolabs Ipswich MA Lot#10078130). Following the PacBio Isoseq protocol, first and second strand cDNA were synthesized using NEB dT oligo and the PacBio Template Switching Oligo. For the cDNA amplification 14 cycles of PCR were performed followed by a Pronex bead cleanup (Promega Corporation Madison WI, Lot #NG103A). The amplified and purified cDNA were QCed using the Bioanalyzer DNA 12000 kit
Approximately 300 ng of cDNA was converted into a SMRTbell library using the Iso-Seq Express Kit SMRT Bell Express Template prep kit 2.0 (Pacific Biosciences). This protocol employs bead-based size selection to remove low mass cDNA, specifically using an 86:100 bead-to-sample ratio (Pronex Beads, Promega). Library preparations were performed in technical duplicate. We sequenced libraries using 11 SMRT cells on the Sequel II system using polymerase v2.1 with a loading concentration of 85pM. A 2-h extension and 30-h movie collection time was used for data collection. The “ccs” command from the PacBio SMRTLink suite (SMRTLink version 9) was used to convert raw reads (~22 million) into Circular Consensus Sequence (CCS) reads. CCS reads with a minimum of three full passes and a 99% minimum predicted accuracy (QV20) were kept for further analysis.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Sequel II

Data processing

samples were demultiplexed using Lime
refinement and clustering using Isoseq V3
samples were CPM normalized within DEseq2
Assembly: GRCh38
Supplementary files format and content: tab delimieted CPM counts

Submission date

Feb 06, 2023

Last update date

Mar 01, 2023

Contact name

Charles R Farber

E-mail(s)

crf2s@virginia.edu

Organization name

University of Virginia

Department

Center for Public Health Genomics