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Sample GSM5230313

Query DataSets for GSM5230313

Status

Public on Jun 01, 2022

Title

MBTD1 3xFlag-2xstrep in ZMYND11 KO-clone #2

Sample type

SRA

Source name

K562 from ATCC

Organism

Homo sapiens

Characteristics

cell line: K562
cell type: lymphoblast from chronic myelogenous leukemia (CML)
growth conditions: normal growth conditions
treatment: ZMYND11 KO by CRISPR/Cas9 and insertion of Tag only at the AAVS1 locus by Zinc finger Nuclease.
expressing: MBTD1 3xFlag-2xstrep

Growth protocol

RPMI, 10% NBCS, 1ug/ml Puromycin

Extracted molecule

polyA RNA

Extraction protocol

For long-read RNA-seq, total RNA was extracted with Rneasy mini columns (QIAGEN), TURBO Dnase treated (ThermoFisher Scientific) and poly(A) enriched with NEBNext poly(A) mRNA Magnetic Isolation Module (NEB).
Libraries were prepared according to Oxford Nanopore Technologies' instructions for the ONT SQK-DCS109 kit, with each sample barcoded with the ONT EXP-NBD104 kit. Briefly, poly(A) RNA was reverse-transcribed into cDNA with the Maxima H Minus RT (ThermoFisher Scientific) and second strand synthesis was accomplished using LongAmp Taq polymerase (NEB). The blunt DNA ends were prepared using the NEBNext Ultra II End Repair/dA-Tailing module (NEB), adding a 3' dA tail and phosphorylating the 5' end. Barcodes were ligated to the DNA fragments with a Blunt/TA ligase (NEB) and motor protein adapters were finally ligated with the NEBNext Quick Ligation Module (NEB). Purification of DNA between each step was performed with KAPA Hyperpure Beads (Roche). Libraries were sequenced on ONT's MinION Mk1B using four R9 flowcells.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

MinION

Data processing

For ChIP-seq, the single-end reads were mapped on the human genome build hg19 using bowtie version 2.1.6 (Langmead et al., 2009), using the pre-set sensitive parameter.
MACS version 2.0.10 (model-based analysis of ChIPseq) peak finding algorithm was used to identify regions of ChIP-seq enrichment over the background (Feng et al., 2012), using broad peaks parameters.
Unique mapped read values were normalized to library size. Peaks were annotated as per human genome EnsDb.Hspaiens.v75 - hg19
For long-read RNAseq, raw fast5 files were basecalled using Guppy v.4.2.2 using fast basecalling settings, and samples were demultiplexed according to barcodes with the guppy_barcoder.
The resulting FASTQ reads were aligned to the GRCh38 human reference with Minimap2 version 2.17-r941
Raw read counts were obtained with the featureCounts tool from the Subread package v 2.0.0, using the exon counting mode (Li, 2018; Liao et al., 2014) and normalized as Counts per Million (CPM).
Genome_build: hg19
Supplementary_files_format_and_content: MBTD1_Flag.IgG.broad_peaks.broadPeak is a bed file containing significant MACS-called broad peaks for the MBTD1 3xFlag-2xstrep ChIP sample using the IgG control sample as input sample.
Supplementary_files_format_and_content: WTZMYND11_Flag.IgG.broad_peaks.broadPeak is a bed file containing significant MACS-called broad peaks for the full length (WT) ZMYND11 3xFlag-2xstrep ChIP sample using the IgG control sample as input sample.
Supplementary_files_format_and_content: trZMYND_Flag.IgG.broad_peaks.broadPeak is a bed file containing significant MACS-called broad peaks for the truncated ZMYND11 [aa1-409] 3xFlag-2xstrep ChIP sample using the IgG control sample as input sample.
Supplementary_files_format_and_content: Fusion_Flag.IgG.broad_peaks.broadPeak is a bed file containing significant MACS-called broad peaks for the ZMYND11-MBTD1 fusion 3xFlag-2xstrep ChIP sample using the IgG control sample as input sample.
Supplementary_files_format_and_content: JC_MD_exp.genes.cpm.txt is a tab separated text file containing read counts for genes normalized to CPM for all long-read RNA-seq samples.
Supplementary_files_format_and_content: JC_MD_exp.transcripts.cpm.txt is a tab separated text file containing read counts for transcripts normalized to CPM for all long-read RNA-seq samples.

Submission date

Apr 08, 2021

Last update date

Jun 01, 2022

Contact name

Samer M.I. Hussein

E-mail(s)

samer.hussein@fmed.ulaval.ca

Organization name

CHU de Québec - Université Laval

Department

Departement of medecine