|
| Status |
Public on Jul 05, 2017 |
| Title |
mu_CD_IL6RaNKdel_PGAT replicate 1 |
| Sample type |
SRA |
| |
|
| Source name |
PGAT
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
diet: CD
organ: PGAT
il6ra deletion in nk cells (del or flox): del
|
| Treatment protocol |
Organs and / or cells were harvested and isolated after completion of the diet periods of time indicated in the manuscript.
|
| Growth protocol |
Diet induced obesity model: Mice were set on ad libitum high-fat-diet (HFD) or normal control diet (CD).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
NK cell subpopulations from murine organs and blood or human blood were purified from single cell suspensions using FACSAria-III or FACSAria-Fusion cell sorters (BD Bioscience) after immunostainings as described above. In mice, NK cells were sorted from obese versus lean animals identified as single, viable CD45+ CD3- NK1.1+ CD11b+ mature NK cells. Final sorting gates of human NK cells identified IL6Ra+ versus IL6Ra- CD3- CD56+ single cells. Purified cells were directly sorted into RNA-protect cell reagent (Qiagen, Germany). Stabilized, purified NK cell populations were pelleted by centrifugation and total RNA was extracted using the Arcturus RNA picopure Kit (KIT0204, ThermoFisher Scientific) following the manufacturer´s instructions. RNA integrity was assessed with the Agilent 2100 Bioanalyzer.
For organs and bulk sorted NK cells: RNA libraries were prepared from a minimum of 100ng total RNA using the TruSeq® RNA sample preparation Kit v2 (Illumina). Complementary DNA (cDNA) was transcribed from poly-A selected RNA, which served for library generation. Libraries were sequenced in replicates for 30 million reads on an Illumina HiSeq 2000 sequencer with a paired-end (101x7x101 cycles) protocol.
Single cells were sorted in 384-well plates containing 240nL of primer mix and 1.2uL of PCR encapsulation barrier, Vapor-Lock (Qiagen GmbH, Germany). Single cell RNA sequencing was performed using CEL-Seq2 method (Hashimshony et al., 2016) with several modifications. Importantly, a fivefold volume reduction was achieved using a nanoliter-scale pipetting robot, Mosquito HTS (TTP Labtech). Sorted plates were centrifuged at 2200g for 10min at 4°C, snap-froze in liquid nitrogen and stored at -80°C until processed. 160nL of reverse transcription reaction mix and 2.2μl of second strand reaction mix was used to convert RNA into cDNA. cDNA from 96-cells was pooled together before clean up and in vitro transcription, generating 4 libraries from one 384-well plate. 0.8μl of AMPure/RNAClean XP beads (Beckman Coulter GmbH, Germany) per 1μl of sample were used during all the purification steps including library cleanup. 8 libraries (768 single cells) were sequenced in a single lane (pair-end multiplexing run, Read 1 30bp Read 2 120bp read length) of Illumina NextSeq 500 sequencing system generating 200 million sequence fragments.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
FPKM_RUN1666.xlsx
IL6Ra_delNK_VAT_CD_1
|
| Data processing |
Illumina Casava software used for basecalling
RNAseq reads were aligned homo_sapiens and mus_musculus
Fragments Per Kilobase of exon per Million mapped fragments (FPKM) were calculated using a Cufflinks
Genome_build: human genome build 38.p10 and mm10
Supplementary_files_format_and_content: excel files
Single cell Data
Read 2 of each read pair was first 3' trimmed for adapters, base quality and poly-A tails using cutadapt 1.9.1 (http://dx.doi.org/10.14806/ej.17.1.200). Remaining sequences were mapped to the mouse genome GRCm38 (primary assembly) using STAR-2.5.2b (https://www.ncbi.nlm.nih.gov/pubmed/23104886). Gene models were used according to gencode version M9 (https://www.ncbi.nlm.nih.gov/pubmed/26187010). Gene summarization was done using featureCounts 1.5.0-p1 (https://www.ncbi.nlm.nih.gov/pubmed/24227677) collapsing exons to genes and excluding pseudogenes and transcripts with a biotype related to "decay". Multimapping reads were discarded.
Cell and gene demultiplexing was done using the cell-barcode and the unique molecular identifier (UMI) present in the first 12nt of Read 1 of the read pair. Libraries L1/L3/L5/L7 use cell barcodes 1-96 and libraries L2/L4/L6/L9 use cell barcodes 97-192. Only exact matches were counted.
Data analysis was performed using RaceID2 and StemID algorithm (Grün et al., 2016). Downsampling to 800 transcripts was used for data normalization. K-medoids clustering was performed using log-pearson correlation as a distance metric. The minimum suitable cluster number (=3) characterizing the dataset was determined by computing Jaccard’s similarity for each cluster by bootstrapping for k-medoids clustering with different cluster numbers. The minimum number yielding a Jaccard’s similarity >0.6 for all clusters was selected. The t-distributed stochastic neighbor embedding (t-SNE) algorithm was used for dimensional reduction and cell cluster visualization (Maaten and Hinton, 2008). RaceID2 was executed with the probability threshold value for outlier identification set to <10 -3. The StemID algorithm was used to infer a dedifferentiation trajectory. A p-value threshold of 0.05 was chosen to assign significance to the links.
Genome_build: mm10
Supplementary_files_format_and_content: Tab-separated text file with transcript counts per gene (corrected for UMI saturation effects according to Grün, Kester and van Oudenaarden (2014) ), text file with cell barcodes
|
| |
|
| Submission date |
May 18, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Sebastian Theurich |
| E-mail(s) |
sebastian.theurich@uk-koeln.de
|
| Organization name |
Max-Planck-Institute for Metabolism Research and University Hospital of Cologne
|
| Lab |
Neuronal Control of Metabolism (Brüning Lab)
|
| Street address |
Gleueler Str. 50
|
| City |
Köln (Cologne) |
| ZIP/Postal code |
50937 |
| Country |
Germany |
| |
|
| Platform ID |
GPL13112 |
| Series (1) |
| GSE99068 |
IL-6/Stat3-Dependent Induction of Distinct, Obesity-Associated Natural Killer Cells Deteriorates Energy and Glucose Homeostasis |
|
| Relations |
| BioSample |
SAMN07141109 |
| SRA |
SRX2834021 |
