|Public on Jan 27, 2021
|Bone marrow Lin- Sca1+ Kit+ cells
|cell types: Mouse-derived Bone marrow cells
tissue: Bone marrow
cell type: Lin- Sca1+ Kit+ cells
|Immediately extract RNA from LSK cells without any treatment
|Total bone marrow cells were taken from WT and Hoxblinc-Tg mice, LSK cells were then sorted using Lin-, Sca1+ and Kit+ markers, and LK cells were sorted using Lin- and Kit+ markers.
|RNA samples extracted with Trizon and treated with Dnase I. ChIP Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with H3K4me3 antibody (antibody: Anti-H3K4me3, Millipore, Cat No. 04-745). ATAC-seq samples derived according to Nextera Tn5 Transposase kits. 4C seq library samples derived from two enzymes digestion BglII and NlaIII and ligation with T4 ligase.
RNA-seq libraries were prepared according to TruSeq Stranded mRNA Library Prep (#20020594). ATAC-seq libraries were prepared according to Nextera DNA Library Prep Kit (#FC-121-1030). 4C-seq libraries were prepared according to TruSeq ChIP Library Preparation Kit (#IP-202-1012). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
|Illumina NextSeq 500
|Standard Illumina software base-calling and quality-control filtering was applied Sequences (Paired end)
Paired end RNA-seq reads were aligned to the mm9 genome assembly using Tophat, and ATAC-seq, ChIP-seq and 4C-seq reads were aligned to the mm9 genome assembly using BOWTIE2 default parameters.
Peak calling was performed using MACS algorithm (version 2.1.1)
Genome coverage tracks were created using deepTools version 3.0
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for RNA-seq Samples and peaks for ATAC-seq, ChIP-seq and 4C-seq
|May 30, 2018
|Last update date
|Jan 27, 2021
|Penn State University
|500 University Dr.
|HOXBLINC is aberrantly expressed in acute myeloid leukemia and functions as a potent oncogenic long non-coding RNA in leukemogenesis