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Sample GSM2858647

Query DataSets for GSM2858647

Status

Public on Mar 29, 2018

Title

GMP_39

Sample type

SRA

Source name

Bone marrow cells

Organism

Mus musculus

Characteristics

strain: C57BL/6
cell population: GMP
sorting strategy: Live Lin- cKit+ Sca1- CD34+ CD16/32hi cells falling outside the BMCP gate

Extracted molecule

polyA RNA

Extraction protocol

Single cells were sorted into 96 well PCR plates containing 2.3 µl lysis buffer consisting of 2.3 U SUPERase In™ RNase Inhibitor (ThermoFisher Scientific, AM2696) in 0.2 % Triton per well. Cells were processed for RNA-sequencing following Picelli et al (Nature Protocols 9, 171-181 (2014)). Briefly, oligo-dT was annealed to polyA-mRNA at 72 °C for 3 minutes, followed by cDNA synthesis using SuperScript™ II Reverse Transcriptase (Invitrogen 18064014). PCR was performed with 23 PCR cycles and products cleaned up using 1:0.6 ratio of cDNA: AMPure XP beads (Beckman Coulter). ERCC Spike In Mix was included in each single cell reaction at 1: 3 000 000 dilution (ThermoFisher Scientific 4456740). Single cell cDNA library was eluted in 20 µl volume of EB Buffer (Qiagen).
Prior to library preparation, cDNA library was checked on Agilent Bioanalyser 2100 with Agilent High Sensitivity DNA Kit. Library preparation was performed using Illumina Nextera XT DNA Library Preparation Kit with 1.26 µl cDNA library and Nextera XT Index Kit v2 Set A and D. Single cell libraries were pooled for all indexed samples and cleaned with AMPure XP beads; first with 1:0.5 ratio of cDNA: beads then the supernatant cleaned with 1:0.2 ratio of cDNA: beads. Pooled libraries were quantified with KAPA Library Quant Kit (KAPA Biosystems KK4824) and single-end 50 base pair sequencing was performed on the Illumina HiSeq 4000 platform.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Data processing

RNA-seq reads were aligned to mm10 using GSNAP (version 2015-09-29) with parameters (-B 5 -t 24 -n 1 -Q -N 1).
Reads in features were counted with htseq-count (HTSeq version 0.5.3p3) with the parameter (-s no).
Genome_build: mm10
Supplementary_files_format_and_content: HT-Seq counts(.txt)
Supplementary_files_format_and_content: extra_sequences.fa: Fasta file contains the sequences used for the customized index.

Submission date

Nov 16, 2017

Last update date

May 15, 2019

Contact name

Evangelia Diamanti

E-mail(s)

[email protected]

Phone

01223 62317

Organization name

University of Cambridge