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Sample GSM4725610

Query DataSets for GSM4725610

Status

Public on Aug 13, 2020

Title

bulkRNAseq_m6ATracer_rep1

Sample type

SRA

Source name

HEK293T cells

Organism

Homo sapiens

Characteristics

cell line: HEK293T
transfection: m6A-Tracer

Treatment protocol

Human HEK293T cells were transiently transfected with Dam-LMNB1 plus m6A-Tracer using FuGene HD, induced using 1 mM Shield-1 ligand for 20 hours prior to harvesting as a single-cell suspension.

Growth protocol

HEK293T cells (CRL-3216, ATCC, Manassas, VA; validated by microsatellite typing & mycoplasma tested, at passage number <10) were cultured in DMEM plus 10%FBS and 1X Penicillin-Streptomycin at 37C with 5% CO2.

Extracted molecule

polyA RNA

Extraction protocol

Cells were processed within nanoliter reaction chambers on a custom PDMS microfluidic device, following the reagents and incubation times of the in-tube single-cell DamID protocol (Kind et al. 2015) with minor modifications (addition of 0.2% Tween-20 to prevent enzyme adhesion). Amplified DNA from each individual cell was flushed out of the device and transferred to 0.2 ml tubes, where library preparation took place.
Single-cell sequencing libraries were prepared from the DamID amplified DNA using an NEBnext UltraII DNA Library Prep Kit for Illumina (samples 001-018) or an NEBnext UltraII FS Kit (samples A01-D10).

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina NovaSeq 6000

Description

17
HEK_mTracer_RNA_1
RNAseq_Counts_GeneBySample.csv

Data processing

Library strategy: DamID
Bulk and single-cell DamID reads were demultiplexed using Illumina’s BaseSpace platform to obtain fastq files for each sample.
DamID and Illumina adapter sequences were trimmed off using trimmomatic (v0.39; Bolger et al. 2014; ILLUMINACLIP:adapters-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:20, where adapters-PE.fa is:\n>PrefixPE/1\nTACACTCTTTCCCTACACGACGCTCTTCCGATCT\n>PrefixPE/2\nGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT\n>Dam\nGGTCGCGGCCGAGGA\n>Dam_rc\nTCCTCGGCCGCGACC).
Trimmed reads were aligned to a custom reference (hg38 reference sequence plus the Dam-LMNB1 and m6A-Tracer plasmid sequences) using BWA-MEM (v0.7.15-r1140, Li 2013). Alignments with mapping quality 0 were discarded using samtools (v1.9, Li et al. 2009).
The hg38 reference sequence was split into simulated DpnI digestion fragments by reporting all intervals between GATC sites (excluding the GATC sites themselves), yielding 7180359 possible DpnI fragments across the 24 chromosome assemblies. The number of reads overlapping each fragment was counted using bedtools (v2.28; Quinlan et al. 2010). For single-cell data, the number of DpnI fragments with non-zero coverage was reported within each non-overlapping bin in the genome (28163 total 100 kb bins, after excluding unmappable regions with zero coverage in any cell).
For bulk data, the number of read pairs overlapping each 100 kb bin was reported. For bulk data, Dam-LMNB1 vs DamOnly enrichment was computed using Deseq2 in each 100 kb bin (Love et al. 2014). For single-cell data, the expected background coverage in each bin was computed as n(m/t), where n is the number of unique fragments sequenced from that cell, m is the number of bulk Dam-only read pairs mapping to that bin, and t is the total number of mapped bulk Dam-only read pairs. Single-cell normalization was computed either as a ratio of observed to expected coverage (for browser visualization and comparison to bulk data), or as their difference (for classification and coverage distribution plotting).
For bulk RNA-seq data, adapters were trimmed using trimmomatic (v0.39; Bolger et al. 2014; ILLUMINACLIP:adapters-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36, where adapters-PE.fa is:\n>PrefixPE/1\nTACACTCTTTCCCTACACGACGCTCTTCCGATCT\n>PrefixPE/2\nGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT). Transcript quantification was performed using Salmon (Patro et al. 2017) with the GRCh38 transcript reference. Differential expression analysis was performed using the voom function in limma (Ritchie et al. 2015). Differential expression was called based on logFC significantly greater than 1 and adjusted p-value < 0.01.
Genome_build: hg38 (GRCh38)
Supplementary_files_format_and_content: hg38_100kb_UniqueFragmentCounts.tsv
Supplementary_files_format_and_content: DamMutantComparison_Counts.tsv
Supplementary_files_format_and_content: RNAseq_Counts_GeneBySample.csv

Submission date

Aug 12, 2020

Last update date

Aug 13, 2020

Contact name

Nicolas Altemose

Organization name

University of California, Berkeley

Department

Bioengineering

Lab

Aaron Streets