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| Status |
Public on Oct 22, 2023 |
| Title |
NPC MAF1 KD RPC1 ChIP-Seq rep1 |
| Sample type |
SRA |
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| Source name |
kucg-2 hiPSC
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| Organism |
Homo sapiens |
| Characteristics |
cell line: kucg-2 hiPSC
cell type: Human neural progenitor
chip antibody: RPC1 (Cell Signaling Technology, #12825)
treatment: MAF1 KD CRISPRi
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed with 0.8% methanol-free formaldehyde in DMEM for 10 min, followed by quenching with 0.125 M glycine for 5 min. Cells were resuspended in Farnham buffer (5 mM PIPES, pH 8; 85 mM KCl; 0.5% IGEPAL-CA 630), followed by snap freezing in liquid nitrogen. All buffers were supplemented with cOmplete™ EDTA-free Protease Inhibitor Cocktail before use. Chromatin was isolated and sheared following the NEXSON protocol. Frozen cell pellets were thawed on ice and sonicated for 2 min in 1- ml tubes on a Covaris S220 with the following settings: peak power = 75W; duty factor = 2%; cycles/burst = 200. Isolated nuclei were washed with Farnham buffer once and resuspended in shearing buffer (10 mM Tris-HCl, pH 8; 0.1% SDS; 1 mM EDTA). Chromatin was sheared on a Covaris S220 by sonication for 18 min in 1-ml tubes with the following settings: peak power = 140W; duty factor = 5%; cycles/burst = 200. Sheared chromatin was snap frozen in liquid nitrogen, aliquoted and stored at -80°C.
Sequencing libraries from ChIP eluted DNA samples were prepared with the Ovation® Ultralow V2 DNA-Seq Library Preparation Kit (TECAN, #0344NB) and SPRIselect beads (Beckman Coulter, #B23318) according to the manufacturer’s protocol. 110-bp paired-end sequencing was performed on a NovaSeq platform (Illumina).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
ChIP-Seq datasets were preprocessed to remove potential 3’ adapters using Trim Galore v0.6.4 with default settings, retaining reads of length ≥20.
Adapter-trimmed ChIP-Seq libraries were aligned to the GRCh38 reference genome using STAR with the following settings: up to one mismatch per read, a maximum of ten alignment positions, end-to-end alignment, prohibited introns, only one alignment reported per read (--outFilterMismatchNmax 1 --outFilterMultimapNmax 10 --alignEndsType EndToEnd --alignIntronMax 1 --outSAMmultNmax 1). Reads from spike-in-containing libraries were also aligned to the D. melanogaster r6.39 genome with the same settings, except only uniquely-mapped reads were retained (--outFilterMultimapNmax 1). Read duplicates were then removed using Picard Tools MarkDuplicates v2.17.10. Peaks were called with MACS callpeak v2.2.6, supplying ChIP input samples from HPSI0214i-kucg_2 for kucg-2 hiPSC and CM datasets, HPSI0214i-wibj_2 for wibj_2 hiPSC datasets, and from HPSI0214i-kucg_2-derived NPC for NPC and neuron datasets, specifying the fragment sizes (--extsize) with shifting model building disabled (--nomodel). The small region size used to calculate dynamic lambda was reduced to 500bp (--slocal 500), and peak summits were also reported (--call-summits). Additionally, for all peak calling, signal normalized per million reads was saved in bedgraph format for visual inspection of datasets (-B and -SMPR). Finally, predicted peaks were filtered using the ENCODE project unified GRCh38 blacklist regions bed file (https://www.encodeproject.org/files/ENCFF356LFX/) by identifying overlaps using bedtools intersect v2.29.2. Blacklist-filtered peak region summits were then annotated by searching for their nearest predicted tRNA locus with bedtools closest Peaks with tRNA “hits” were then defined for each sample as those within 125 bp of an annotated tRNA gene, while tRNA hits shared by both replicates of each cell type or experimental condition were used to define consensus tRNA peaks for that condition.
Assembly: GRCh38
Supplementary files format and content: narrowPeak, broadPeak: Peaks called with macs2 (except input samples)
Supplementary files format and content: control lambda bedGraph for (Input control samples only)
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| Submission date |
Mar 22, 2023 |
| Last update date |
Oct 22, 2023 |
| Contact name |
Danny Nedialkova |
| E-mail(s) |
nedialkova@biochem.mpg.de
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| Organization name |
Max Planck Institute for Biochemistry
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| Lab |
Mechanisms of Protein Biogenesis
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| Street address |
Am Klopferspitz 18
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| City |
Planegg |
| State/province |
Bayern |
| ZIP/Postal code |
82152 |
| Country |
Germany |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE227924 |
Transfer RNA pools in human cells are controlled by selective gene expression [ChIP-seq] |
| GSE227928 |
Selective gene expression maintains human tRNA anticodon pools during differentiation |
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| Relations |
| BioSample |
SAMN33861883 |
| SRA |
SRX19748717 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7111044_MAF1_KD_RPC1_k_NPC_rep1.narrowPeak.gz |
30.8 Kb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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