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| Status |
Public on Nov 02, 2022 |
| Title |
RBG18806_Old_AEC_001 |
| Sample type |
SRA |
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| Source name |
endosteum and central marrow
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| Organism |
Mus musculus |
| Characteristics |
cell population: mesenchymal and endothelial cells
cell_type: AEC
age: Old
icgscluster_ec: 3
icgscluster_ec_merged: gEC2-3
icgscluster_nonec: NA
icgscluster_nonec_merged: NA
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| Extracted molecule |
total RNA |
| Extraction protocol |
Single endosteal and central marrow stromal cells were directly sorted into individual wells of a 96-well PCR plate containing 2.3 µl of 0.2% Triton-X100 (Sigma-Aldrich, 93443) and 1U of Superase-In RNase Inhibitor (Ambion). Of note, information regarding expression of surface markers was recorded for each cell. Cells were frozen immediately at -80C until further processing. After thawing on ice, 2 µl of annealing mixture (1 µM oligodT –IDT-, 5mM each dNTPs and a dilution 1:6,000,000 of ERCC RNA Spike-In Mix -Invitrogen-) was added followed by incubation at 72C for 3 minutes. Then 5.7 µl of Reverse Transcription mix (3.5 U/µl of Maxima H minus retrotranscriptase –ThermoFisher-, 0.88 U/µl of Superase-In RNase Inhibitor, 1.75X Maxima RT Buffer, 3.5 µM TSO – Qiagen- and 13.15% PEG 8000 –Sigma-Aldrich-) was added and the mixture was incubated at 42 C for 90 min, followed by 70 C for 15 min. cDNA was further amplified by adding 40 µl of PCR mix (0.03 U/µl of Terra PCR direct polymerase –Takara Bio-, 1.25X Terra PCR Direct Buffer, 0.25 µM IS PCR primer –IDT-). PCR was as follows: 3 min at 98 °C for initial denaturation followed by 21 cycles of 15 s at 98 °C, 30 s at 65 °C, 4 min at 68 °C. Final elongation was performed for 10 min at 72 °C. Sequences of oligodT, TSO and IS PCR primers were as previously described (PMID: 24385147).
Following preamplification, all samples were purified using Ampure XP beads (Beckman Coulter) at a ratio of 1:0.6 with a final elution in 25 µl of EB Buffer (Qiagen). The cDNA was then quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher). Size distributions were checked on high-sensitivity DNA chips (Agilent Bioanalyzer). Samples were used to construct Nextera XT libraries (Illumina) from 100 pg of preamplified cDNA. Libraries were purified and size selected (0.5X-0.7X) using Ampure XP beads. Then, libraries were quantified using KAPA qPCR quantification kit (KAPA Biosystems), pooled and sequenced in an Illumina HiSeq 4000 instrument.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Reads were mapped to the Mus musculus genome (EMSEMBL GRCm38.p4 Release 81) and ERCC sequences using GSNAP (version 2015-09-29) with parameters: -A sam –B 5 –t 24 –n 1 –Q –N 1.
HTseq-count (PMID: 25260700) was used to count reads mapped to each gene, with parameters: –s no.
Genome_build: GRCm38.p4
Supplementary_files_format_and_content: HT-Seq counts / gene counts (.txt)
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| Submission date |
Mar 10, 2021 |
| Last update date |
Nov 02, 2022 |
| Contact name |
Bertie Gottgens |
| Organization name |
University of Cambridge
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| Department |
Haematology
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| Street address |
Hills Road
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| City |
Cambridge |
| ZIP/Postal code |
CB2 0XY |
| Country |
United Kingdom |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE168613 |
Inflammation of the Aged Bone Marrow Niche Drives Blood Aging |
| GSE169162 |
Stromal niche inflammation mediated by IL-1 signaling is a targetable driver of hematopoietic aging |
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| Relations |
| BioSample |
SAMN18238855 |
| SRA |
SRX10302402 |
| 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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