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Status |
Public on Jul 23, 2017 |
Title |
KO-CONTROL 155 |
Sample type |
SRA |
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Source name |
10-mo-old IFNAR-KO 14 days following AAV-CTRL administration
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Organism |
Mus musculus |
Characteristics |
strain: mic-IFNAR-KO organ: brain selection marker: CD11b+ CD45int treatment: AAV-CTRL
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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 NextSeq 500 |
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Description |
Supplementary_table_4.txt
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Data processing |
Illumina bcl2fastq software used for basecalling. Sample barcodes were extracted from read 2 and concatenated to the fastq header of read 1. Barcode is of size 7 followed by UMI of size 8 alignment: hisat 0.1.5 with deafult parameters filter PCR amplification bias using alignment break site and UMI barcode (size 8). create tag directory using makeTagDirectory from HOMER tools (http://homer.salk.edu/homer/ngs/) HOMER, quantifying RNA expression in genes: analyzeRepeats.pl rna mm9 -d <List of tag dir> -count 3utr -condenseGenes -strand + -norm 1e7 Genome_build: mm9 Supplementary_files_format_and_content: tab-delimited text files include mRNA molecule count values for each Sample
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Submission date |
May 01, 2017 |
Last update date |
May 15, 2019 |
Contact name |
Ido Amit |
E-mail(s) |
ido.amit@weizmann.ac.il
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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 |
GPL19057 |
Series (1) |
GSE98401 |
MEF2C restrains microglial responses to inflammatory stimuli and is reduced upon IFN-I in brain aging |
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Relations |
BioSample |
SAMN06855454 |
SRA |
SRX2772023 |
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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