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| Status |
Public on Feb 10, 2023 |
| Title |
ATAC-seq for C9HRE B lymphocytes from a ALS/FTD patient |
| Sample type |
SRA |
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| Source name |
B lymphocytes
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| Organism |
Homo sapiens |
| Characteristics |
cell type: B lymphocytes
genotype: C9orf72 HRE mutation
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| Growth protocol |
human B lymphocytes were cultured in RPMI 1640 medium containing 15% FBS. Medium was exchanged every other day.
Human iPSCs were maintained in StemFlex medium (Gibco, A3349401), with medium exchange every other day.
Motor neuron differentiation: human iPSCs were seeded onto Matrigel-coated plates and differentiated into neuroepithelial progenitor cells (NEPCs) by culturing 6 days in neural medium (1:1 DMEM/F12:neurobasal medium, GlutaMax, N2 supplement, B27 supplement, and ascorbic acid) containing 3 μM CHIR99021, 2 μM SB431542, and 2 μM DMH-1. NEPCs were dissociated with dispase (1 U/ml) and split into new plates coated with Matrigel with a ratio of about 1:6. NEPCs were differentiated into motor neuron progenitor cells (MNPCs) using neural medium supplemented with 1 μM CHIR99021, 2 μM SB431542, 2 μM DMH-1, 0.1 μM retinoic acid (RA), and 0.5 μM purmorphamine. After culturing for 6 days, MNPCS were dissociated and cultured in suspension using neural medium containing 0.5 μM RA and 0.1 μM purmorphamine. Six days later, neural spheres were dissociated and plated onto a Matrigel-coated plate. Adherent neural spheres were cultured in neural medium supplemented with 0.5 μM RA, 0.1 μM purmorphamine, and 0.1 μM compound E. After 12 days, mature motor neurons were acquired for experiments. The medium was changed every other day during the entire differentiation period.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Human B lymphocytes (~1×105) or progenitor-differentiated motor neurons (~1.6×106) were used for ATAC-seq. Cells were pelleted in 1× PBS (500 g for 5 min, 4°C) and resuspended in 50 μl of cold lysis buffer (10 mM Tris-HCl [pH 7.4], 10 mM NaCl, 3 mM MgCl2, and 0.1% IGEPAL CA-630) with gentle pipetting. Cell lysates were centrifuged (500×g for 10 min, 4°C) to pellet the nuclei, which were then incubated with 50 μl of transposition reaction buffer (25 μl of 2× Tagment DNA reaction buffer [Illumina FC-121-1030], 2.5 μl of Nextera Tn5 Transposase [Illumina FC-121-1030], and 22.5 μl of nuclease-free H2O) at 37°C for 30 min with gentle rotation. After the reaction, genomic DNA was purified with a Qiagen MinElute PCR Purification Kit (Qiagen, 28204) and eluted in 10 μl of elution buffer (10 mM Tris buffer [pH 8.0]).
To amplify the transposed DNA fragments, the DNA sample was mixed with PCR amplification buffer containing nuclease-free H2O, Nextera PCR Primer 1, Nextera PCR Primer 2 (barcode), and NEBNext High-Fidelity 2× PCR Master Mix (New England Labs, M0541), and then amplified by 1 cycle of 72°C incubation for 5 min and 98°C for 30 sec, followed by 5 cycles of 98°C for 10 sec, 63°C for 30 sec, and 72°C for 1 min. To avoid PCR saturation of a library and reduce the GC and size bias, 5 μl of the first PCR reaction were amplified in a side qPCR reaction to select an appropriate cycle number for the second-round PCR reaction, which was set to 1 cycle of 98°C incubation for 30 sec, followed by 20 cycles of 98°C for 10 sec, 63°C for 30 sec, and 72°C for 1 min. The cycle number corresponding to a quarter of the maximum fluorescence intensity was used in the final PCR amplification. After the final amplification, PCR products were purified using a Qiagen MinElute PCR Purification Kit (Cat. NO. 28204) and analyzed by deep sequencing.
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Adapters were removed using Cutadapt
The quality of the reads was measured by FastQC.
The trimmed reads were sequentially mapped to the human reference genome (hg39 version) using Bowtie in an end-to-end model.
Duplicate fragments were removed with Picard MarkDuplicates.
MACS2 was used to find peak callings.
Assembly: hg39
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| Submission date |
Jan 19, 2023 |
| Last update date |
Feb 10, 2023 |
| Contact name |
Yang Liu |
| E-mail(s) |
liuyang19870210@outlook.com
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| Organization name |
Johns Hopkins University
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| Department |
Biochemistry and Molecular Biology
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| Lab |
Wang Lab
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| Street address |
615 N. Wolfe Street
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| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21211 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE223255 |
ATAC-Seq analysis of the chromatin accessibilities in C9HRE ALS/FTD patient cells |
| GSE223259 |
Chromatin accessibilities in C9HRE ALS/FTD patient cells |
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| Relations |
| BioSample |
SAMN32798272 |
| SRA |
SRX19076286 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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