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Sample GSM6098468

Query DataSets for GSM6098468

Status

Public on May 02, 2024

Title

HSC-D6

Sample type

SRA

Source name

HSCs purified from challenged wild-type C57BL/6J mice with Listeria monocytogenes (LM) at 3 days after the secondary LM challenge。

Organism

Mus musculus

Characteristics

cell type: HSCs
strain: C57BL/6
tissue: bone marrow
age: 6-8 weeks
genotype: wild type

Extracted molecule

total RNA

Extraction protocol

In brief, intact bone marrows from mice at the age of 6-8 weeks were flushed from mouse femora and tibiae. Bone marrow cells by pipetting with 1 mL syringe, and then underwent red blood cell lysis using 0.16 M ammonium chloride solution.
Low input RNA seq was performed according to published scRNA seq protocols with modifications (Picelli et al., 2014). Briefly, FACS-sorted 1500 Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) (3-5 µL) of each sample were transferred into 40 µL cell lysis buffer prepared by adding 2 µL of RNase inhibitor to 38 µL of a 0.25% (v/v) Triton X-100 solution. Quickly vortex the tube to mix, and then purify RNA using 80 µL VAHTS RNA Clean Beads (N412, Vayzme), following reverse transcription with 5 µL purified RNA and 15 µL RT mix of each sample. cDNA purified with VAHTS DNA Clean Beads (N411, Vazyme) was used to perform PCR enrichment with 13 cycles of amplification. Enriched cDNA was quantified using Qubit dsDNA HS Assay Kit (Q3285, Guangzhou IGE biotechnology ltd) after purification. TruePrepTM DNA library Prep Kit V2 (TD503, Vazyme) was used to prepare RNA seq library following the manufacturer’s protocol. Libraries were sequenced as paired 150 bp using NovaSeq-PE150.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

RNA seq reads were aligned to mouse genome GRCm38 and gene annotation from the Ensembl database. For read alignment and expression quantification, low quality reads were removed and the adaptor sequences were trimmed by Trim Galore (v0.5.0, Babraham Bioinformatics). The remaining pair-end reads were mapped to the reference genome by Salmon (v0.03.1, COMBINE-lab) with ENCODE options. The uniquely mapped reads were counted and transformed to transcripts per kilobase millionreads (TPM)
Assembly: GRCm38
Supplementary files format and content: ABCD_edgeR_CPM_annotated.csv with raw counts matrices

Submission date

May 03, 2022

Last update date

May 02, 2024

Contact name

chen minqi

E-mail(s)

chenmq57@mail2.sysu.edu.cn

Phone

+8617307510403

Organization name

Sun Yat-sen University