GSM4698441: bone marrow sorted fixed cells H3K27me3; Mus musculus; OTHER
Mouse leg 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. DNA fragments are further blunt ended using Klenow large fragment and T4 PNK in the presence of dNTPs. Subsequently blunt fragments are A-Tailed using AmpliTaq 360 DNA Polymerase. Next fragments are ligated to T-tail adaptors containing 3 bp UMIs and an 8bp cell barcode using T4-ligase. Ligation products were pooled by centrifugation and cleaned. The cleaned DNA is then linear amplified by invitro transcription. The produced RNA is purified, followed by DNAse1 digestion and aRNA fragmentation. aRNA is reverse transcribed. Single stranded DNA is purified through RNAseA incubation followed by PCR amplification to add the Illumina smallRNA barcodes and handles.
GEO Accession
GSM4698441