NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM3855292 Query DataSets for GSM3855292
Status Public on May 10, 2024
Title B6_BM-H3K9me3-m1-1_AH5W5KBGXB_S9
Sample type SRA
 
Source name B6_BM
Organism Mus musculus
Characteristics strain: B6
age: 13weeks
tissue: bone marrow
antibody: H3K9me3
Extracted molecule genomic DNA
Extraction protocol Femur and Tibia were extracted, the bones ends were cut away to access the bone marrow which was flushed out using a 22G syringe with HBSS (-ca, -mag, -phenol red, Gibco 14175053) supplemented with Pen-Strep and 1% FCS. The bone marrow was dissociated and debris was removed by passing it through a 70um cell strainer (Corning, 431751). Cells were washed with 25ml supplemented HBSS before depleting the sample of unnucleated cells using IOTest 3 Lysing solution (Beckman Coulter) following provider instructions. Cells were washed additional 2 times with PBS before processing them by the scChIC protocol for the 4 different histone modifications.
Cells were resuspended in 500ul wash buffer (47.5ml H2O RNAse free, 1ml 1M HEPES pH 7.5, 1.5ml 5M NaCl, 3.6ul pure spermidine solution). Aliquot in a 0.5ml protein low-binding tubes (1 per antibody to be used). Following steps are performed on ice. Cells were pelleted at 600g for 3min and resuspended in 400 ul Wash Buffer 1 (wash buffer with extra 0.05% saponin (Sigma Aldrich), protease inhibitor cocktail (Sigma Aldrich), 4 ul/ml 0.5M EDTA) containing the primary antibody (1:100 dilution for H3K4me1, H3K9me3 and H3K27me3 and 1:200 dilution for H3K4me3, Saponin has to be prepared fresh every time as a 10% solution in PBS). Cells were incubated overnight at 4oC on a roller, before they were washed once with 500 ul Wash Buffer 2 (wash buffer with extra 0.05% saponin, protease inhibitor). Afterwards cells were resuspended in 500 ul Wash Buffer 2 containing PaMN (3ng/ml) and incubated for 1h at 4C on a roller. Finally, cells were washed an additional 2 times with 500 ul Wash Buffer 2 before passing it through a 70um cell strainer (Corning, 431751) and sorting them on a JAZZ FACS machine into 384 well plates containing 50nl Wash buffer 3 (Wash buffer containing 0,05% saponin) and 5ul sterile filtered mineral oil (Sigma Aldrich) per well. Small volumes were distributed using a Nanodrop II system (Innovadyme). 50nl of Wash Buffer 3, containing 4uM CaCl2, were added per cell to induce Protein A-MN mediated chromatin digestion that was performed for 30min on ice. Afterwards the reaction was stopped by adding 100nl of a stop solution containing 40mM EGTA (chelates Ca2+ and stops MN), 1.5% NP40 and 10ul 2mg/ml proteinase k (Ambion). Chromatin is subsequently released and PaMN permanently destroyed by proteinase k digestion at 65C for 6h followed by 80C for 20min to heat inactivate proteinase k. Afterwards plates can be stored at -20C until further processing.
DNA fragments are further blunt ended using Klenow large fragment (NEB) and T4 PNK (NEB) in the presence of dNTPs. Afterwards nonintegrated dNTPs are removed using schrimp alkaline phosphatase (NEB) to increase A-tail efficiency. Subsequently blunt fragments are A-Tailed using AmpliTaqâ„¢ 360 DNA Polymerase (Thermofisher) in the presence of fresh dATP and T4 PNK (NEB). Next fragments are ligated to T-tail adaptors containing 3 bp UMIs and an 8bp cell barcode (final concentration 9nM from IDT) for 16h at 16C using T4-ligase (NEB). Adaptors were added with a Mosquito HTS (ttp labtech). Ligation products were pooled by centrifugation and cleaned using Ampure XP beads at a bead to sample ratio of 0.8 (Beckman Coulter). The cleaned DNA is then linear amplified by invitro transcription with components of MessageAmp II (Invitrogen) for 16h at 37C. The produced RNA is purified 1-3 times depending on Adaptor dimer contamination using RNA Clean XP beads (Beckman Coulter) at 0.8 beads to sample ratio, followed by RNA fragmentation for 2.5 min at 94C. After another bead cleanup, 20% of the RNA is reverse transcribed with Superscript II (NEB) and PCR amplified to add the Illumina smallRNA barcodes and handles with NEBNext Ultra II Q5 Master Mix. PCR cycles depended on the abundance of the histone modification assayed and the number of RNA cleanup cycles after IVT needed (8-10 for H3K9me3 and H3K27me3 10-12 for H3K4me1 and 12-15 for H3K4me3).
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina NextSeq 500
 
Description genomicDNA_cut_from_MNase-ProteinA_with_staining_of_H3K9me3
B6-13W1-BM-H3K9me3-4-merged.filtered.bincounts.slidewin.csv.gz
Data processing fastq files were mapped using BWA-0.7.16a (r1181) to ENSEMBL Build 95
Cells in the top 95% of UMI counts and fraction counts starting with "TA" are kept as "good cells".
Bins were created using 100 kb sliding window with step size of 20 kb.
Genome_build: mm10
Supplementary_files_format_and_content: Genomic bins to cells binned across genomic regions.
 
Submission date Jun 05, 2019
Last update date May 10, 2024
Contact name Jake Yeung
E-mail(s) jakeyeung@gmail.com
Organization name Hubrecht Institute
Street address Uppsalalaan 8
City Utrecht
ZIP/Postal code 3584T
Country Netherlands
 
Platform ID GPL19057
Series (1)
GSE132266 scChIC-seq reveals focusing of global chromatin domains during differentiation
Relations
BioSample SAMN11963310
SRA SRX5978581

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap