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Sample GSM2871546

Query DataSets for GSM2871546

Status

Public on Aug 03, 2018

Title

Differentiated Ctrl sgRNA 1

Sample type

SRA

Source name

U937 cell line

Organism

Homo sapiens

Characteristics

protocol: Differentiated Ctrl sgRNA
cell line: U937

Treatment protocol

For differentiation, U937 cells were diluted to a density of 1-2 million cells per mL in U937 growth medium containing phorbol myristate acetate (PMA) to achieve a final concentration of 50 nM PMA. Cells were plated on tissue culture plastic and allowed to differentiate and adhere for 3 days. After differentiation, cells were recovered by harvesting supernatants and trypsinizing adherent cells. For trypsinization, cells were washed three times with phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 8 mM sodium phosphate dibasic, 2 mM potassium phosphate monobasic, pH 7.4), incubated in PBS containing 300 units/mL trypsin for 7 min at 37°C, then recovered from the plate by repeatedly pipetting the solution to dislodge cells. Trypsinized adherent and non-adherent supernatant cells were pooled, pelleted by centrifugation (300g, 7 min, room temperature), and resuspended in fresh U937 growth medium. Cells were then plated and allowed to recover for two days before further processing.

Growth protocol

U937 cells were acquired from ATCC (CRL-1593.2). Cells were maintained in suspension culture using spinner flasks for library propagation and tissue culture plates for single-gene knockout lines, all in sterile-filtered U937 growth medium (RPMI1640 supplemented with 2 mM glutamine, 100 units/mL penicillin, 100 mg/mL streptomycin, and 10% heat-inactivated fetal calf serum). Cells were cultured in a humidified 37°C incubator set at 5% CO2. Cells were passaged 2-3 times weekly.

Extracted molecule

total RNA

Extraction protocol

Differentiated and undifferentiated U937 cells were seeded at a density of 50,000 cells per well in a 24-well plate. Two days later, cells were lysed in RLT buffer and RNA was isolated using the RNeasy Micro Kit (Qiagen).
cDNA libraries were prepared and adapted for sequencing using a modified Smart-seq2 protocol (Picelli et al., 2014). In short, 10 ng of purified RNA was mixed with RNase Inhibitor, Oligo-dT30 primer, and dNTPs and heat-denatured before addition to a SuperScriptII template-switching reverse transcription reaction mixture. cDNA was amplified in a 12-cycle KAPA HiFi HotStart PCR reaction and purified using Ampure XP SPRI beads. 0.5 ng of cDNA was fragmented for sequencing using the Nextera XT DNA sample preparation kit. After tagmentaion and amplification of adapter-ligated, indexed fragments, DNA was purified using Ampure XP SPRI beads and sequenced on a NextSeq 550 (Illumina) to obtain 75 bp paired-end reads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

NextSeq 550

Description

D_CTRL_1

Data processing

RNA-seq data was mapped to the human genome annotation hg38 using HISAT2 version 2.0.3 (Kim et al., 2015) via the Galaxy platform (https://usegalaxy.org/) (Afgan et al., 2016), resulting in 8 to 12 million mapped reads per sample. Transcript FPKM (fragments per kilobase of transcript sequence per million mapped fragments) scores were obtained using Cufflinks version 2.2.1 (Trapnell et al., 2010) with the iGenomes hg38 gtf file as a reference annotation.
The counts and FPKM tables do not contain processed data for D_GFP_1. This was omitted due to a processing error and was left out of the analysis.
Genome_build: hg38
Supplementary_files_format_and_content: CBMH2_Counts_GEO.xlsx and CBMH2_FPKM_GEO.xlsx contain counts from DESeq2 and FPKM values from Cufflinks respectively

Submission date

Dec 01, 2017

Last update date

May 15, 2019

Contact name

Christopher John Bohlen

E-mail(s)

[email protected]

Organization name

Genentech

Department

Neuroscience