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Sample GSM5239175 Query DataSets for GSM5239175
Status Public on Nov 28, 2021
Title Liver_WT_DD_CT16_B
Sample type SRA
 
Source name Liver_WT_DD_CT16
Organism Mus musculus
Characteristics genotype: WT
strain: C57BL/6
time point: CT16
tissue: Liver
Treatment protocol Mice were kept in constant darkness, and harvested in 4 h intervals, starting at CT0 (beginning of first subjective day) until CT44 h.
Growth protocol 3mo mice were housed under standard light-dark conditions (12h/12h) for 2 weeks before the experiment, in standard conditions with food provided ad libitum.
Extracted molecule total RNA
Extraction protocol Tissues were snap-frozen in liquid nitrogen immediately after dissection and stored at -80°C till used. For RNA extraction, the tissues were soaked in TRI-reagent (Sigma) and were homogenized by bead beater (Bead Ruptor e24, OMNI), and then proceeded by a standard TRI-reagent based RNA extraction protocol. RNA concentration was determined using NanoDrop™ 2000 Spectrophotometer (Thermo Fisher Scientific). RNA Integrity was validated using 2200 TapeStation (Agilent).
cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500.
3' RNA-seq for digital gene expression quantitation
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model NextSeq 550
 
Description processed data file: R370_Liver_WT_reads.xlsx
Data processing Illumina bcl2fastq software used for basecalling.
The rest of analysis was done through the UTAP pipline. Reads were trimmed using cutadapt
Reads were mapped to genome (/shareDB/iGenomes/Mus_musculus/UCSC/mm10/Sequence/STAR_index) using STAR (default parameters)
The pipeline quantifies the genes annotated in RefSeq (that have expanded with 1000 bases toward 5’ edge and 100 bases toward 3’ bases)
Counting was done using htseq-count (union mode)
Normalization of the counts was performed using DESeq2 with the parameters: betaPrior=True, cooksCutoff=FALSE, independentFiltering=FALSE
Genome_build: mm10
Supplementary_files_format_and_content: xlsx files containing for each gene and sample the raw counts and normalized counts
 
Submission date Apr 13, 2021
Last update date Nov 28, 2021
Contact name Gal Manella
Organization name Weizmann Institute of Science
Street address 234 Herzel St.
City Rehovot
State/province Israel
ZIP/Postal code 7610001
Country Israel
 
Platform ID GPL21626
Series (1)
GSE171975 Ultradian Molecular Rhythms Emerge in the Absence of a Circadian Clock
Relations
BioSample SAMN18721860
SRA SRX10592158

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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