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Sample GSM4222114

Query DataSets for GSM4222114

Status

Public on Jul 31, 2021

Title

3M_Dup_clone_4_rep_2

Sample type

SRA

Source name

3M cerebral organoids

Organism

Homo sapiens

Characteristics

tissue: 3M cerebral organoids
16p11.2 cnv: DUPLICATION
disease state: autism

Growth protocol

iPSCs: To generate induced pluripotent stem cells (iPSCs), skin fibroblasts were infected with Sendai virus vectors containing coding sequences of human OCT4, SOX2, KLF4 and c-MYC (Cytotune reprogramming kit, Thermo Fisher). Four days post infection, fibroblasts were trypsinized to single cells, plated on the inactivated mouse embryonic fibroblast feeders, and cultured using human embryonic stem cell medium (vendor?). After 3–4 weeks, iPSC clones were manually picked and propagated clonally on feeders. After 8-10 passages, iPSCs were transferred to a feeder-free system and grown on matrigel-coated dishes (Corning) in mTeSR1 media (StemCell Technologies). The cells were passaged by manually picking colonies.
Cerebral organoids: Feeder-free iPSCs at passage 15 or later were fed daily with mTeSR1 for at least 7 days before differentiation. Colonies were dissociated using Accutase (Life Technologies) in PBS (1:1) for 10 minutes at 37°C and centrifuged for 3 minutes at 100xg. The cell pellet was resuspended in mTeSR1 supplemented with 10 μM SB431542 (SB, Tocris) and 1 μM Dorsomorphin (Dorso, R&D Systems). Approximately 5 × 106 cells were transferred to each well of a 6-well plate and kept in suspension under rotation (95 rpm) in the presence of 5 μM ROCK inhibitor (StemCell Technologies) for 24 hours to form free-floating spheres. Then, the media was replaced with mTeSR1 for additional 48 hours. After 72 hours, Media1 [Neurobasal (Life Technologies) supplemented with Glutamax, 2% Gem21 NeuroPlex (Gemini Bio-Products), 1% N2 NeuroPlex (Gemini Bio-Products), 1% MEM nonessential amino acids (NEAA, Life Technologies), 1% penicillin/streptomycin (PS; LifeTechnologies), 10 μM SB and 1 μM Dorso] was used for maintenance for 7 days, with media changes every other day. Subsequently, Media1 was replaced with Media2 [Neurobasal with Glutamax, 2% Gem21 NeuroPlex, 1% NEAA and 1% PS] supplemented with 20 ng/mL FGF2 (Life Technologies) for additional 7 days. Then, Media2 was supplemented with both 20 ng/mL FGF2 and 20 ng/mL EGF (Life Technologies) and spheres were cultured for additional 7 days with media changes every other day. Next, organoids were transferred into Media3 [Media2 supplemented with 10 ng/mL BDNF, 10 ng/mL GDNF, 10 ng/mL NT-3 (all from Life Technologies), 200 μM L-ascorbic acid (Tocris) and 1 mM dibutyryl-cAMP (StemCell Technologies)] for another 7 days with media changes every other day. After 28 days, cortical organoids were maintained in Media2 for as long as needed, with media changes every 3-4 days. All organoids were generated, grown and used for all experiments in the same plate with one DEL, one DUP and one CTRL (called a “batch” thereafter) to reduce batch effect from genotypes.

Extracted molecule

total RNA

Extraction protocol

RNA-seq libraries were prepared using standard Illumina protocols

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Data processing

Illumina RTA version 2.7.6 software used for basecalling.
All fastqs were trimmed for adapter sequence and low base call quality (Phred score < 30 at ends) using Cutadapt (v1.14).
Trimmed reads were then aligned to the GRCH37.p13 (hg19) reference genome via STAR (2.5.3a) using comprehensive gene annotations from Gencode (v19).
Gene-level quantifications were calculated using RSEM (v1.3).
Genome_build: Human GRCH37.p13 (hg19) reference genome
Supplementary_files_format_and_content: count matrix with rows as genes (EnsembIDs) and columns as samples

Submission date

Dec 17, 2019

Last update date

Jul 31, 2021

Contact name

Lilia M Iakoucheva

E-mail(s)

lilyak@health.ucsd.edu

Phone

(858)-822-1878

Organization name

University of California San Diego