|
| Status |
Public on Apr 08, 2016 |
| Title |
100pgTechRepUMI_primer [UMI_primer_sample_0013] |
| Sample type |
SRA |
| |
|
| Source name |
total RNA
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: N2
tissue: embryos
age: mixed stages
experimental design: 100pg of C. elegans isloted total RNA was used to calibrate the new CEL-Seq2 protocol. Each sample set represent different parameters; old_bc - the original CEL-Seq primer, UMI - unique molecular identifier, short - a shorter version of the original primer with UMI, no_lig - ligation free 3 prime illumina adapter introduction.
|
| Growth protocol |
Mouse ear fibroblasts were derived from 1 month-old CyclinB1-GFP mice. Briefly, a small piece of mouse ear was collected, digested overnight in DMEM containing Collagenase /Dispase. The tissue was then dissociated by gentle pipetting and cells were washed in DMEM/10% FBS/1% Pen-Strep/1% L-Glutamine, pelleted, seeded and cultured for 2-4 days before sorting on a FACS ARIA (Becton Dickinson).
Bone marrow dendritic cells (BMDCs) were collected from 6-8 weeks old female C57BL/6J mice from femora and tibiae and plated. At day 9 post-bone marrow extraction non-adherent cells were collected and stained for CD11c to collect BMDCs. day 5, floating CD11c+ cells were sorted on the
|
| Extracted molecule |
total RNA |
| Extraction protocol |
C. elegans total RNA was isolated using TRIzol with addition of ERCC spike-ins and GenElute LPA (Sigma). BMDCs were lysed by sorting directly into a hypotonic solution before freezing on dry ice. Mouse fibroblasts were lysed in lysis buffer 0.2% NP-40 on C1 IFC. Manually collected fibroblasts were lysed by freezing in liquid nitrogen.
For RNA amplification and library construction see the detailed protocol in "CEL-Seq2: sensitive highly-multiplexed single-cell RNA-Seq"
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
100pgTechRepUMI_primer.txt
UMI_primer_sample_0013
|
| Data processing |
Libraries were sequenced on the Illumina HiSeq2500 according to standard, paired-end sequencing, protocols. Primary analysis done in RTA 1.17.20,1.13.48
Conversion from BCL to FASTQ and deumltiplexing using CASAVA-1.8 (configureBclToFastq.pl--fastq-cluster-count 1234567890 --mismatches 0 --use-bases-mask Y15n,I6n,Y35n)
Filter and read trimming (barcode minimum quality of 20. trimming of read2 to 35 bases - not required).
CEL-Seq (Hashimshony, et al. 2012) demultiplexing of second mate, using first mate barcode, allowing no mismatches in barcode.
bowtie2, version 2.1.0, against various genomes
Read counting with htseq-count version 0.5.4p3. Reads with the same unique molecular identifiers (UMIs) were colapsed for transcript counting.
Genome_build: GRCm38
Genome_build: mm9 for comparison to Smart-Seq data
Genome_build: WS230
Supplementary_files_format_and_content: Tabular expression matrix includes number of transcript molecules.
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| |
|
| Submission date |
Mar 01, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Naftalie Senderovich |
| E-mail(s) |
naftalys@tx.technion.ac.il
|
| Organization name |
Technion - Israel Institute of Technology
|
| Department |
Biology
|
| Lab |
Itai Yanai
|
| Street address |
Technion city, Faculty of biology
|
| City |
Haifa |
| State/province |
International |
| ZIP/Postal code |
3200003 |
| Country |
Israel |
| |
|
| Platform ID |
GPL18245 |
| Series (1) |
| GSE78779 |
CEL-Seq2: sensitive highly-multiplexed single-cell RNA-Seq |
|
| Relations |
| BioSample |
SAMN04525026 |
| SRA |
SRX1606371 |
