GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM5255146

Query DataSets for GSM5255146

Status

Public on Mar 16, 2022

Title

Exp2_2D_LRRK050401-01_1

Sample type

SRA

Source name

dopaminergic neurons

Organism

Homo sapiens

Characteristics

cell type: hiPSC-derived dopaminergic neurons
genotype: LRRK2 G2019S
parkinson's disease (pd) status: PD
culture condition: Two dimensional (2D)
experiment number: 2

Treatment protocol

On day 31, cells were pre-differentiated DA neurons were seeded into 2D and 3D culture condition

Growth protocol

On day 0, a single cell suspension of hiPSCs (maintained with mTeSR1) was seeded in 50:50 (v/v) mTeSR1:floor plate specification medium (neurobasal medium (ThermoFisher 21103049) with 1X floor plate specification supplement (ThermoFisher A3146801)) supplemented with 10 μM Y-27632 (STEMCELL Technologies 72302) at a density of 2 x 10^5 cells/cm2 on 6-well plates coated with Matrigel. The next day, cells were washed with Dulbecco’s phosphate buffered saline (DPBS, ThermoFisher) and fresh specification medium was added. Medium was carefully changed every 48 hours. On day 11, floor plate progenitors (FPPs) were split 1:2 using Accutase (Innovative Cell Technologies AT104, San Diego, CA, USA) onto new Matrigel-coated 6-well plates with FPP expansion medium (ThermoFisher A3165801) + 10 μm Y-27642. FPPs were expanded (replacing FPP expansion medium every 48 hours) until cryopreservation on day 22 with Synth-a-Freeze reagent (ThermoFisher A1254201) to establish batches of cells for experimental consistency. For each individual experiment, FPPs were thawed and plated at a density of 6.25 x 10^5 cells/cm2 in Matrigel coated 6-well plates supplemented with 10 μM Y-27642. On day 28, FPPs were differentiated to DA neurons starting with a 72 hour treatment of maturation medium (DMEM/F12 (ThermoFisher 10565018) with 1X maturation supplement (ThermoFisher A3147401)) containing 1 μM PD0325901 (MilliporeSigma PZ0162, Burlington, MA, USA) and 5 μM SU5402 (MilliporeSigma SML0443). PD0325901 and SU5402 were employed to inhibit progenitor cell proliferation and promote neurogenesis.

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated from independent cultures using the RNeasy mini kit (Qiagen 74106, Hilden, Germany) utilizing the on-column DNA-digestion procedure according to manufacturer’s instructions. To improve extraction efficiency, the 2D cultures and 3D constructs were gently sonicated in the kit’s lysis buffer prior to extraction (ThermoFisher model no. FB120, 20% power, 1 second on followed by 1 second off for 10 pulses total). Sample purity and concentration was measured with a Nanodrop (ThermoFisher) spectrophotometer and 1 μg RNA per sample with a 260/280 nm reading greater than 2 were sent to Novogene for additional quality control tests, library preparation, and sequencing. First, RNA degradation and contamination were assessed on 1% agarose gels and RNA purity was checked using a NanoPhotometer spectrophotometer (Implen, Munich, Germany). RNA integrity and quantitation were assessed using the RNA Nano 6000 assay kit of the Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA).
Library preparation was achieved using the NEBNext Ultra RNA library prep kit for Illumina systems following manufacturer’s recommendations (New England Biolabs, Ipswich, MA, USA) and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand complementary DNA (cDNA) was synthesized using random hexamer primer and M-MuLV reverse transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptors with a hairpin loop structure were ligated to prepare for hybridization. To select cDNA fragments of 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Brea, CA, USA). Then 3 μL USER Enzyme (New England Biolabs) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 minutes followed by 5 minutes at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system, Beckman Coulter) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on an Illumina Novaseq sequencer according to the manufacturer’s instructions. After cluster generation, the libraries were sequenced on the same machine and paired-end reads were generated at a read depth of 20 million reads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

Illumina pipeline CASAVA v1.8.2 was used for base calling.
Raw sequencing data was uploaded to the Tufts University Galaxy Server for quality and adapter trimming with Trim Galore, read alignment with RNA STAR (reference human genome build 38), and read quantification with FeatureCounts using default settings.
Normalized read count tables, obtained by dividing the read counts by their size factors, were generated and extracted with the DESeq2 R package.
Genome_build: reference human genome build 38
Supplementary_files_format_and_content: Matrix table with normalized gene counts for every gene and every sample

Submission date

Apr 20, 2021

Last update date

Mar 16, 2022

Contact name

Nicholas James Fiore

E-mail(s)

[email protected]

Organization name

Tufts University

Department

Biomedical Engineering