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| Status |
Public on Dec 05, 2014 |
| Title |
b04_Kupffer_Exp2 |
| Sample type |
SRA |
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| Source name |
Kupffer cells
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
tissue: Liver
cell type: Kupffer cells
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
10^4-10^5 cells from each macrophage subpopulation were sorted in 100-200 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C
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
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 1500 |
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| Data processing |
Illumina Casava1.8 software used for basecalling.
alignment: tophat with deafult parameters
Normalized expression values are produced using ESAT software available at http://garberlab.umassmed.edu/software/esat/
Genome_build: mm9
Supplementary_files_format_and_content: mRNA molecule count values for each Sample
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| Submission date |
Nov 17, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Ido Amit |
| E-mail(s) |
[email protected]
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| Phone |
972-8-9343338
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| Organization name |
Weizmann Institute of Science
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| Department |
Immunology
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| Street address |
234 Herzl st.
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| City |
Rehovot |
| ZIP/Postal code |
760001 |
| Country |
Israel |
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| Platform ID |
GPL18480 |
| Series (2) |
| GSE63340 |
Tissue-resident macrophage enhancer landscapes are shaped by the local microenvironment [RNA-seq] |
| GSE63341 |
Tissue-resident macrophage enhancer landscapes are shaped by the local microenvironment |
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| Relations |
| Reanalyzed by |
GSE80797 |
| BioSample |
SAMN03196821 |
| SRA |
SRX760626 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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