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Sample GSM4310176 Query DataSets for GSM4310176
Status Public on Dec 31, 2020
Title Sample_4su_PPM1G_KO_2_S3
Sample type SRA
Source name Sample_4su_PPM1G_KO
Organism Homo sapiens
Characteristics cell line: HCT116
cell type: Colorectal Carcinoma Cell Line
genotype/variation: PPM1G KO
passages: Under 15
molecule subtype: nascent 4sU RNA
Growth protocol All HCT116 cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum and 1% Pen/Strep at 37°C with 5% CO2.
Extracted molecule total RNA
Extraction protocol For 4sU-seq, HCT116 Ctrl and PPM1G KO cells were seeded into 6-well dishes per biological replicate and grown to 70% confluency. Cells were pulsed for 10 min with DMEM + 500 µM 4sU (Sigma-Aldrich, T4509). At the 9 min mark the DMEM + 4sU media was removed and cells were washed one time with 1X PBS. The PBS was removed, and cells were lysed on-plate using the standard TRizol protocol (ThermoFisher Scientific, 15596026). RNA samples were DNase treated (NEB, M0303) and then extracted with acid:phenol chloroform (ThermoFisher Scientific, AM9720) followed by an additional chloroform extraction. RNA was then precipitated with 1/10 volume of NaCl (5M), 1 µL glycoblue, and 2.5 volumes of 100% EtOH. RNA pellets were washed with 1 mL of 75% EtOH and air dried and then resuspended in RNase-free water. RNA (300 µg) was brought to a final concentration of 0.4 mg/mL with RNase-free water. A separate aqueous master mix (20 mM NaOAC pH 5.2, 1 mM EDTA pH 8.0, 0.1% SDS) was added to the diluted RNA mix followed by the addition of 0.2 mg/mL Biotin-HPDP (ThermoFisher Scientific 21341) in dimethylformamide. The biotinylation reaction was incubated at room temperature for 2 hrs followed by acid:phenol chloroform extraction. Biotinylated RNA pellets were resuspended in 500 µL RNA Prurification Beads (RPB) buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA pH 8.0, 300 mM NaCl). Dyna MyOne Strepavidin T1 beads (200 uL/sample; ThermoFisher Scientific, 65601) were blocked at room temperature in beads wash buffer + 1% polyvinylpyrrolidone for 10 min. After blocking, beads were washed 1X with 1 mL beads wash buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA pH 8.0, 50 mM NaCl) and then resuspended in RPB buffer. Biotinylated, 4sU RNA was denatured at 65°C for 5 min and then placed on ice for 2 min. 200 µL of blocked beads were added to each sample and incubated for 30 min with rotation at room temperature. Beads were washed 5X with 4sU wash buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA pH 8.0, 1 M NaCl, 0.1% Tween-20). Biotinylated, 4sU RNA was eluted from beads 2 times in 75 µL of 0.1 M DTT for 15 min at room temperature with rotation, giving a final elution volume of 150 µL. 4sU-RNA was further purified using the Zymo RNA Clean and Concentrator Kit (Zymo Reasearch, R1013). RNA concentration was measured using the Qubit RNA high sensitivity assay (ThermoFisher Scientific, Q32852) and quality was determined using the Agilent Tapestation RNA ScreenTape (Agilent, 5067-5576). For RNA-seq, HCT116 Ctrl and PPM1G KO cells were grown in 6-well dishes and total RNA extracted using the Zymo Quick-RNA Miniprep Kit (R1055) according to manufacturer instructions.
For 4sU-seq and RNA-seq, the KAPA Stranded RNA-seq kit with RiboErase (HMR) (KAPA Biosystems, KK8483) was used to prepare libraries according to manufacturer’s instructions. Briefly, 200 ng of 4sU RNA and 1 µg of total RNA was used to prepare 4sU-seq and RNA-seq libraries, respectively. Libraries were rRNA depleted followed by DNase treatment. RNA was fragmented and primed followed by 1st strand cDNA synthesis. 2nd strand synthesis was then performed with the incorporation of dUTP. Libraries were then A-tailed, adapter ligated, and amplified. All libraries were quantified using the Qubit 2.0 fluorometer and library size verified using the Agilent Tapestation DNA D1000 ScreenTape (Agilent, 5067-5582). All libraries were submitted to the McDermott Center Sequencing Core at UT Southwestern. 75 bp paired-end reads were generated for all libraries.
Stranded Whole Transcriptome RNA-seq
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
Description ERCC spike-in RNA added
processed data file: 4sUseq_countTable.txt
Data processing Quality of reads from all experiments was determined with FASTQC v0.11.2 using default parameters.
4sU-/RNA-seq: Reads were aligned to hg38 with hisat2 version 2.1.0-intel.
4sU-/RNA-seq: Sorted by coordinate BAM files were created by SAMtools v1.3.
4sU-/RNA-seq: ERCC spike-ins were similarly aligned to an ERCC fasta file.
4sU-/RNA-seq: Reads for protein coding genes were counted using the Gencode v27 annotation and featureCounts.
4sU-/RNA-seq: Differential expression analysis was performed using RUVseq v1.14.0 R package.
Genome_build: hg38
Supplementary_files_format_and_content: 4sU-/RNA-seq: Bigwig files were scaled by the ERCC spike-in
Supplementary_files_format_and_content: 4sU-/RNA-seq: Tab-delimited file containing raw transcript counts
Submission date Feb 13, 2020
Last update date Jan 01, 2021
Contact name Ivan D'Orso
Phone 214-633-1374
Organization name UT Southwestern Medical Center
Department Microbiology
Lab NL3.110A
Street address 5323 Harry Hines
City Dallas
State/province TX
ZIP/Postal code 75390-9048
Country USA
Platform ID GPL18573
Series (1)
GSE145249 Transcriptional reprogramming of cancer cells upon loss of the PPM1G/PP2Cγ phosphatase
BioSample SAMN14096299
SRA SRX7719015

Supplementary file Size Download File type/resource 86.0 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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