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Sample GSM6634281

Query DataSets for GSM6634281

Status

Public on Oct 14, 2023

Title

HL1_short-read_nucleoplasmfraction_rep2

Sample type

SRA

Source name

HL1

Organism

Mus musculus

Characteristics

cell line: HL1
rna fraction: chromatin

Growth protocol

HL1 mouse cardiomyocytes were grown per manufacturer instructions in supplemented Claycomb Medium (51800C-500ML) + 10% FBS (TMS-016) + 0.1mM Norepinephrine with 30mM L-ascorbic acid (A0937, A7506) + 5% L-Glutamine (G7513) in 0.1% gelatin-coated (SF008) T75 flasks. To prepare the cells for RNA harvesting, they were washed once with 1X PBS, detached with TrypLE Express (Gibco #12605010), quenched with FBE-containing media, then washed with PBS, and pelleted by centrifugation for 5 min at 250 x g

Extracted molecule

nuclear RNA

Extraction protocol

For nuclear fractionation and RNA isolation, cell pellets were resuspended in Buffer A (10 mM HEPES•KOH pH 7.5, 10 mM KCl, 10% glycerol, 340 mM sucrose, 4mM MgCl2, 1 mM DTT, 0.1% Triton X-100, 1 x Protease Inhibitor Cocktail (PIC) [1mM AEBSF, 0.8 μM aprotinin, 20 μM leupeptin, 15 μM pepstatin A, 40 μM bestatin, 15 μM E-64; from 200x DMSO stock]) to obtain nuclei. Purified nuclei were extracted with 0.5 M Urea and 0.5% NP-40 substitute to solubilize loosely bound factors from chromatin and fractionated by centrifugation (Werner, 2015;Werner, 2017). The supernatant was termed as the nucleoplasm fraction and the pellet as the chromatin fraction. 1 μL of a 1:10 diluted ERCC RNA Spike-in standards aliquot (Life Technologies 4456740) was added to each nuclear fraction prior to addition of TRIzol reagent (Life Technologies 15596026). RNA from each fraction was processed using Zymo RNA Clean & Concentrator kit (Zymo Research R1017) with on-column DNase digestion. Ribosomal RNA was removed using Ribo-zero Gold rRNA depletion kit (Illumina MRZG12324) for HL1 short-read RNA seq samples and RiboMinus kit (Invitrogen A15026) for HL1 long-read samples.
For short-read RNA-seq, 100 ng rRNA-depleted RNA from each nuclear fraction was used with the NEBNext Ultra II Directional library kit (NEB E7765S) to prepare RNA-seq libraries. RNA-seq libraries of nuclear fractions (chromatin extract and nucleoplasm extract) from three independent cultures were sequenced on the Illumina HiSeq 4000 to obtain 50bp single end reads. For long-read RNA-seq, two HAP1 long-read sequencing libraries were generated as follows. rRNA-depleted RNA was polyadenylated with E. coli Poly(A) Polymerase (M0276S) (for later priming with long read sequencing primers) and precipitated with ethanol. Polyadenylated chromatin fraction RNA was subjected to library preparation according to the Oxford Nanopore direct cDNA sequencing kit protocol (SQK:DCS109). 10-50 fmol of library was loaded onto a MinION (MIN-101B) flow cell (R9.4.1; FLO-MIN106D) and run for 48 hours.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

HL1_short-read_nucleoplasmfraction_rep2
HL1_short-read_DESeq2_normalizedgenecounts.csv

Data processing

For short-read RNA-seq, read quality and adapter content was checked using FastQC (Andrews, 2015). Reads were then aligned to reference genomes (mm10) catenated with ERCC sequences using hisat2 version 2.1.0 (Kim, 2019) with --rna-strandness R --dta options. Sam files were converted to sorted bam files using samtools (Li, 2009). StringTie (Pertea, 2015) was used to assemble transcripts from sorted bam files to obtain gtf files for each sample. These gtf files were merged using stringtie --merge --rf. This merged transcriptome gtf file was then used as a reference for re-estimation of transcripts in each of the samples using stringtie -e -B -G. Raw counts of transcript and gene abundance were obtained using prepDE.py script from StringTie. Raw counts were then used to calculate differentially expressed transcripts and genes as well as normalized transcript and gene counts using DESeq2. For long-read RNA-seq, real-time basecalling information and FASTQ files were generated with the MinKNOW software using default settings (HL1 rep1: minQ 7; HL1 rep2: minQ 9). FASTQ files generated by the MinKNOW software were concatenated and mapped to mm10+ERCC genomes using minimap2 (Li, 2018) without canonical splice sites (-ax splice -un). After sorting and indexing the BAM files (samtools), coverage analyses were performed using Stringtie2 (Kovaka, 2019) with options -A to generate gene coverage in addition to transcripts and -L to specify long reads. Replicates were then analyzed in tandem using the Stringtie --merge function (-e -B -G) to create a reference GTF with the union of transcripts between both replicates to calculate tags per million (TPM) for transcripts in individual replicates.
Assembly: mm10
Supplementary files format and content: HL1_short-read_DESeq2_normalizedgenecounts.csv is a csv file containing Deseq-normalized gene counts from three biological replicates each of chromatin and nucleoplasm fraction using short-read RNA-seq
Supplementary files format and content: HL1_long-read_StringTie_transcriptabundance.csv is a csv file containing tags per million (TPM) of transcripts in two biological replicates calculated by StringTie in the long-read RNA-seq sample

Submission date

Oct 12, 2022

Last update date

Oct 14, 2023

Contact name

Alexander J Ruthenburg

E-mail(s)

[email protected]

Organization name

University of Chicago

Department

Molecular Genetics and Cell Biology