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Sample GSM1210546

Query DataSets for GSM1210546

Status

Public on Feb 28, 2014

Title

Mut_DRH-3_Rep2

Sample type

SRA

Source name

Mut_DRH-3

Organism

Caenorhabditis elegans

Characteristics

genotype/variation: drh-3 (ne4253) mutant
Stage: late L3/early L4
tissue: whole animal

Growth protocol

Synchronous populations of worms were grown for approximately 40 hr posthatching at 20°C on OP-50 E. coli at a density of ~100,000 animals per 15 cm Petri dish.

Extracted molecule

total RNA

Extraction protocol

Worms were washed off the plate with cold M9 and washed 3-4 times. The worm pellet was resuspended in 2ml of cold Nuclear Run-On (NRO) lysis buffer (10mM Tris pH 7.5, 2mM MgCl2, 3mM CaCl2, 0.5% IGEPAL and 10% glycerol), transferred to a steel dounce (on ice) and stroked 20 times. The worm lysate was centrifuged for 2min at 40g and the supernatant was centrifuged again for 5min at 1000g to pellet the nuclei. Next, the supernatant was discarded and the nuclei pellet was washed two times with 1ml of cold NRO lysis buffer and one time with 1ml of cold Freezing Buffer (50mM Tris-CL pH 8.3, 40% glycerol, 5mM MgCl2, 0.1 mM EDTA). Nuclei were then resuspended in 100µl of Freezing Buffer and used for the NRO reaction. // NRO reaction and DNase digestion: 100µl of nuclei were mixed with an equal volume of reaction buffer (10mM Tris-Cl pH 8.0, 5mM MgCl2, 1mM DTT, 300mM KCL, 20 units of SUPERase In, 1% sarkosyl, 500µM ATP, GTP, and Br-UTP, 2μM CTP) and incubated for 5 minutes at 32°C. The reaction was stopped with 750μl of TRIzol reagent (Invitrogen) and the RNA was extracted following the manufacturer's instructions. The RNA pellet was resuspended in 85µl water and treated with 5µl of turbo DNase (Ambion) in a total volume of 100µl and incubated at 37°C for 30 minutes. The RNA was then extracted by phenol:chloroform and precipitated with one volume of isopropanol with 1/10 volume of Sodium Acetate 3M and 10µg of Glycoblue (Ambion). // Base Hydrolysis of RNA: The hydrolysis of RNA was performed using fragmentation reagent (Ambion) for 15min at 70°C and the product was purified by p-30 RNase free spin column (BioRad), following the manufacturer’s instruction. // Immunopurification of Br-U RNA and RNA-end repair: The purification of the Br-U RNA and the end repair of the purified Br-U RNA was performed as described in (Core et al., 2008).
Adapter ligation and library preparation: For the ligation of 5' and 3' adapters to Br-U RNA we used reagents contained in the ScriptMiner™ Small RNA-sequencing kit (Epicentre) and we immunopurified the ligated Br-U RNA product after each ligation with anti-deoxy-BrU beads (Santa Cruz Biotech (sc-32323-AC). The ligated Br-U RNA was then reverse-transcribed and PCR-amplified with properly-indexed primers for 18 cycles, following the ScriptMiner™ Small RNA-sequencing kit (Epicentre) manufacturer's instructions. The NRO-cDNA libraries were purified by phenol:chloroform, the DNA was precipitated as above and then ran on a non-denaturing 1XTBE, 8% acrylamide gel and stained with SYBR gold (Invitrogen). DNA fragments ranging in size from 120bp to 300bp was excised from the gel and eluted by incubating in TE + 300mM NaCl overnight with rotation. The DNA libraries were then extracted, precipitated, and sent for sequencing.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

GRO-Seq
Replicate 2
processed data file: Mut_DRH-3_rep1+2.neg_strand.bw
processed data file: Mut_DRH-3_rep1+2.pos_strand.bw

Data processing

Two independent biological replicates were generated for csr-1 hypomorphic strain and the corresponding N2 wild type (WT) using ~300,000 worms (called Mut_CSR-1 and WT_CSR-1, respectively). Since drh-3 (ne4253) mutant strain has a reduced number of progeny we performed two biological replicates of drh-3 (ne4253) mutant using ~100,000 worms and we compared them with two biological replicates of N2 (WT) generated using an equal number of worms (Mut_DRH-3 and WT_DRH-3, respectively). Sequencing was performed to a length of 50 nucleotides. Since cloned RNA ranged from 16 to 180 nucleotides in length, the sequencing reads were post-processed to eliminate the 3' linker sequence, if present, so that resulting reads ranged from 16 to 50 nucleotides in length.
The reads were then aligned using “bwa 0.6.1” to the C. elegans genome (ce6 assembly), and rRNA reads were removed based on genomic locations: ChrI:15060000-15072421 for Ce_18S_rDNA; ChrV:17115500-17132700 for Ce_5S_rDNA; chrV:17428800-17429040 for Ce_5S_rDNA-singleton; chrX:16929621-16940441 for Ce_repetitiveLocus. After filtering, we obtained 44,155,515 reads from csr-1 hypomorphic mutant [26,584,972 reads from the first replicate (called Mut_CSR-1_Rep1) and 17,570,543 reads from the second replicate (called Mut_CSR-1_Rep2)] and 36,133,711 reads from the corresponding WT [16,052,870 reads from the first replicate (called WT_CSR-1_Rep1) and 20,080,841 reads from the second replicate (called WT_CSR-1_Rep2)]. From drh-3 (ne4253) mutant, we obtained after filtering 34,588,133 reads [17,492,181 reads from the first replicate (called Mut_DRH-3_Rep1) and 17,095,952 reads from the second replicate (called Mut_DRH-3_Rep2)], and 63,000,071 reads from the corresponding WT [21,660,645 reads from the first replicate (called WT_DRH-3_Rep1) and 41,339,426 reads from the second replicate (called WT_DRH-3_Rep2)].
We further filtered the data by strand (+/-) to analyze sense and antisense Pol II transcription. After ascertaining concordant results with the individual replicate pairs (WT vs. mutant) (data not shown), replicate reads for each WT and mutant were pooled, using downsampling on the replicate with higher read count to ensure equal representation of either replicate, and the pooled reads were processed together to increase statistical power.
For each annotated C. elegans transcript, the aligned reads were binned based on the annotated transcriptional start site (TSS) and transcriptional termination site (TTS) into 3 regions as follows: 'promoter' (from 600bp upstream of the annotated TSS to 200bp downstream), 'gene body' (from 200bp downstream of the annotated TSS to 200bp upstream of the annotated TTS), and 'gene end' (from 200bp upstream of the annotated TTS to 400bp downstream).
We then calculated region-specific RPKM values (#total reads / (region length (KB) X # mapped reads (M)) for these regions. We also calculated statistical significant change in RPKM values using using DESeq package (Anders and Huber, 2010) considering a p value <0.05.
For the enrichment or depletion of gene sets, we first calculated the number of gene expected by chance according to r = (n1xn2)/N, with n1 = number genes in set 1, n2 = number of genes in set 2, N = total number of C. elegans genes considered, and then the enrichment was calculated by dividing the number of genes in common to the two datasets by r. The average data profile in Figure 4B and Figures S4A and S4B were calculated using Cistrome/Galaxy (Liu et al., 2011) with the CEAS tool (SitePro: Aggregation plot tool for signal profiling (version 1.0.0)), and the heatmap showing the genome-wide correlation coefficient values in Figure 4C was calculated using Multiple wiggle files correlation tool (version 1.0.0). The overlaps between gene lists were calculated using the online tool at http://www.nemates.org. Representation factors “r” to quantify the enrichment or depletion of gene sets were calculated according to r = (n1,2)/[(n1xn2)/N], with n1,2 = number of genes common to set 1 and 2, n1 = number genes in set 1, n2 = number of genes in set 2, N = total number of genes considered.
Genome_build: WS190
Supplementary_files_format_and_content: bigWig files, strand-specific pooled replicates, rescaled to sample population size. The files are combined replicates, normalized mapped reads separated by strands.

Submission date

Aug 16, 2013

Last update date

May 15, 2019

Contact name

Germano Cecere

E-mail(s)

gc2352@columbia.edu

Organization name

Columbia University

Department

Biochemistry and Molecular Biophysics