|
| Status |
Public on Nov 10, 2019 |
| Title |
NEO1+Hoxb5+ LT-HSCs [Sample1] |
| Sample type |
SRA |
| |
|
| Source name |
Bone marrow
|
| Organism |
Mus musculus |
| Characteristics |
strain: Hoxb5-mCherry
tissue: bone marrow
age: 2-to-3-month-old
gating strategy: Lin- cKIT+ SCA1+ CD150+ CD48- FLK2- CD34- Hoxb5+ NEO1+
|
| Extracted molecule |
total RNA |
| Extraction protocol |
250-500 cells from two pooled mice per sample were sorted directly into 100 μL of lysis buffer (Buffer RL) and RNA was isolated with the Single Cell RNA Purification Kit (Norgen Biotek Corp.) according to the manufacturer’s protocol. RNA quality was measured by capillary electrophoresis using the Agilent 2100 Bioanalyzer with Nano mRNA assay at the Stanford Protein and Nucleic Acid (PAN) Facility
Libraries were prepared using the Smart-seq2 protocol by Picelli et al., 2014 with minor modifications. Briefly, cDNA was generated by oligo-dT primed reverse transcription with MMLV reverse transcriptase (SMARTScribe, Clontech) and a locked template-switching oligonucleotide (TSO). This was followed by 18 cycles of PCR amplification using KAPA HiFi hotStart ReadyMix and ISPCR primers. Amplified cDNA was then purified using 0.7x volume Agencourt AMPure XP beads to remove smaller fragments. The resulting cDNA concentration and size distribution for each well was determined on a capillary electrophoresis-based Agilent 2100 Bioanalyzer with High Sensitivity DNA chip at the Stanford PAN facility. 40 ng of cDNA was then tagmented, uniquely barcoded, and PCR enriched using the Nextera DNA Library Prep Kit (Illumina, San Diego, CA). Libraries were then pooled in equimolar amounts and purified of smaller fragments using 0.7x Agencourt AMPure XP beads. Pooled libraries were checked for quality using the Agilent Bioanalyzer with High Sensitivity DNA chip at the Stanford PAN facility.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
mRNA
|
| Data processing |
Bcl2fastq2 v2.18 (Illumina) was used to extract the data and generate FASTQ files for each sample by using unique barcode combinations from the Nextera preparation
Raw reads were trimmed for base call quality (PHRED score >=21) and for adapter sequences using Skewer v0.2.2 with default settings
Trimmed reads were then aligned to the mouse genome assembly (mm10) from UCSC (http://genome.ucsc.edu) using STAR v2.4 with default setting
Raw counts from STAR v2.4 were inputted into a DESeqDataSet (DESeq2 v1.22.2 package in R) object indicating NEO1 status (‘status’) and mouse subject (‘subject’) as factors (‘design = ~subject + status’). Counts were then size-factor normalized using the ‘DESeq’ function and log2-transformed.
Genome_build: mm10
Supplementary_files_format_and_content: Tab-delimited text files include (1) raw read count values for each sample (2) size factor- and log-normalized count values for each sample
|
| |
|
| Submission date |
Apr 30, 2019 |
| Last update date |
Nov 10, 2019 |
| Contact name |
Gunsagar Singh Gulati |
| E-mail(s) |
gunsagargulati@stanford.edu
|
| Organization name |
Stanford University
|
| Department |
Institute for Stem Cell Biology and Regenerative Medicine
|
| Lab |
Irving Weissman
|
| Street address |
265 Campus Drive
|
| City |
Palo Alto |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
| |
|
| Platform ID |
GPL19057 |
| Series (1) |
| GSE130504 |
Neogenin-1 distinguishes between myeloid-biased and balanced Hoxb5+ mouse long-term hematopoietic stem cells |
|
| Relations |
| BioSample |
SAMN11538974 |
| SRA |
SRX5771121 |
