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Sample GSM7716902

Query DataSets for GSM7716902

Status

Public on Feb 27, 2024

Title

NGN2_AroPERFECT_12h_r1 TT-Slamseq

Sample type

SRA

Source name

ZIP13K2

Organism

Homo sapiens

Characteristics

cell line: ZIP13K2
treatment: expressing NGN2 AroPERFECT 12h replicate 1

Treatment protocol

To study immediate effects of NGN2 Wild type, AroLITE and AroPERFECT overexpression on ZIP13K2 human iPS cells, cells were treated with doxycycline for 12 or 24 hours and subjected to 15 minutes of 4-Thiouridine (4sU) labeling using 500 µM 4sU

Growth protocol

We adapted our protocol for the differentiation of human iPSCs into neurons by overexpression of NGN2 from 41. ZIP13K2 cells with integrated NGN2 overexpression cassette were cultured on Matrigel (Corning) pre-coated 10 cm culture plates. When cultures reached a confluency of approximately 80%, 2 μg/mL Doxycycline (Sigma) was added to the culture medium to induce expression of the NGN2 transgene. After 24 hours, induced cultures were sorted for mEGFP expressing cells by flow cytometry. Positive cells were seeded on Matrigel pre-coated 96-well microclear plates (Greiner bio-one) in mTeSR+ and 1x Rho-kinase inhibitor at a density of 2x104 cells / cm2. On day 2, mTeSR+ medium was replaced by N2B27 neural cell culture medium supplemented with 5 g/ml human BDNF (biotechne). Differentiation medium was changed every day for a total of 4 days. Living cells were stained with 0.25 g/ml Hoechst and Spy650-TUB (1:2000) (Spirochrome) and incubated in the microscope prior to image acquisition to equilibrate and thermalize all materials

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted with Trizol (Ambion) and 24:1 chloroform:isoamylalcohol (Sigma) while using 0.1 mM dithiothreitol (DTT) in isopropanol precipitation and ethanol washes. For each sample, 50 µg of total RNA was fragmented with Magnesium RNA Fragmentation Module (NEB), and fragmentation buffer was removed from samples with ethanol precipitation in presence of 0.1 mM DTT. RNA was then resuspended in 350 µl RNase-free water, diluted in biotinylation buffer (200 mM HEPES pH 7.5, and 10 mM EDTA) and topped up with 5 µg MTS-Biotin (previously diluted to 50 µg ml–1 in dimethylformamide) to reach a final volume of 500 µl. The biotinylation reaction was incubated for 30 min at room temperature while keeping samples in rotation and protected from light. Unbound biotin was removed with acid-phenol:chloroform extraction (125:24:1, Ambion) and isopropanol precipitation. Biotinylated RNA was resuspended in 100 µl RNase-free water, denatured in 65 °C for 10 min and then cooled on ice for 5 min. The biotinylated RNA was captured with 100 µl µMACS streptavidin beads (Miltenyi) by incubating for 15 min in rotation while keeping samples protected from light. µMACS columns were equilibrated on magnetic stand with nucleic acid equilibration buffer and two times with biotinylation buffer (20 mM HEPES, 1 mM EDTA, pH 8). Beads were transferred to columns and washed three times with wash buffer (100 mM Tris-HCl pH 7.5, 10 mM EDTA, 1 M NaCl and 0.1 % Tween 20), and labeled RNA was eluted twice with a total 200 µl of 100 mM DTT. RNA was cleaned up with RNeasy Minelute columns (Qiagen) and eluted to RNase-free water with 1 mM DTT. 4sU residues of RNA were alkylated with iodoacetamide treatment (10 mM iodoacetamide in 50 mM NaPO4, pH 8 and 50 % DMSO) by incubating samples in 50 °C for 15 min, followed by quenching with 20 mM DTT. RNA samples were purified with ethanol precipitation and treated with Turbo DNase (Invitrogen).
Sequencing libraries were prepared with NEBNext Ultra II Directional RNA Library Prep Kit and NEBNext Multiplex Oligos (NEB), according to manufacturer’s instructions, except using 8 min incubation time in fragmentation ste

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina NovaSeq 6000

Description

TT-Slamseq library of ZIP13K2 cells expressing NGN2 AroPERFECT 12h replicate 1

Data processing

Raw reads were filtered and trimmed as described above for BulkRNAseq samples. Filtered reads were aligned using STAR v2.7.9a with parameters ‘–outFilterMultimapNmax 50–outReadsUnmapped Fastx’ to the SILVA database69 (downloaded 6 March 2020) to remove rRNA content. Unaligned reads were afterwards reverse-complemented using the seqtk ‘seq’ v 1.3-r106 using the ‘-r’ parameter (https://github.com/lh3/seqtk). Reverse-complemented reads were processed using SLAM-DUNK with the ‘all’ pipeline v0.4.1 using the ‘-rl 100 -5 0’ parameters with the GENCODE gene annotation v39 as ‘-b’ option. Reads with a ‘T > C’ conversion representing nascent transcription were filtered from the BAM files using alleyoop with the ‘read-separator’ command. Counts per gene were quantified based on the ‘T > C’-converted reads using featureCounts v 2.0.6 97 using -s 1 and -t gene parameters for stranded and gene body counting.
Supplementary files format and content: NGN2_TTslamseq_Counts_gene.csv has the counts on gene body
Library strategy: TT-Slamseq

Submission date

Aug 17, 2023

Last update date

Feb 27, 2024

Contact name

Alexandre P Magalhaes

E-mail(s)

magalhae@molgen.mpg.de

Organization name

Max Planck Institute for Molecular Genetics

Street address

Ihnestrasse 63-73