|Public on Jun 25, 2018
|Cytosolic Spt5 KD control
|HeLa_Cytosolic Spt5 KD control
|cell line: HeLa
genotype/variation: Spt5 KD
treated with: none
molecule subtype: cytoplasmic RNA
|HeLa cells were transfected with control Spt5 RNAi plasmids along with a plasmid direcing puromycin resistant gene. Transfected cells were selected with puromycin. 72 hours post transfection cell were trearted with TNFα for one hour.
|Hela cells were grown using standard growth conditions
|Cells were separated into cytosol and chromatin fractions which were then subjected to RNA extraction and DNAseI tretment
Cytosolic and chromatin associated RNA samples were depleted of ribosomal RNA using Ribozero gold rRNA depletion kit (Epicentre). RNA was processed using the TruSeq RNA Sample Preparation Kit v2 protocol (Illumina)without poly A capturing. Libraries were evaluated by Qubit and TapeStation. Sequencing libraries were constructed with barcodes to allow multiplexing of 4 samples on one lane. Paired-end 101 bp reads were sequenced on Illumina HiSeq 2500 rapid mode with cBot on HiSeq Rapid Flow Cell v1 HCS: 220.127.116.11.
|Illumina HiSeq 2500
|Demultiplexing and base calling was done with CASAVA 1.8.2 (RTA 18.104.22.168).
Reads were aligned to the hg19 genome built using Tophat (v2.0.04) with an annotation file (refSeq from igenomes UCSC).
Raw gene read counts were calculated by HTSeq using refSeq gene annotations (igenomes).
Normalized gene counts were calculated with DESeq. Chromatin and cytosol samples were analysed separately.
Supplementary_files_format_and_content: Excel file containing gene counts
|Jun 25, 2015
|Last update date
|May 15, 2019
|The Weizmann Institute of Science
|2 Hertzl st.
|RNA-seq of cytosolic and chromatin-associated transcripts following TNFα and Spt5 KD