BioSample

Protocols: Cells were dissociated in Accutase for 5 min and pipetted vigorously to obtain a single cell suspension in normal culture media. Single cells were picked by mouth pipette using a blunted microcapillary with internal diameter 15 μM and under a dissection microscope. Cells were collected into 0.2 mL tubes with 4 μL lysis buffer and immediately snap frozen on dry ice. Cram files were first converted bam and then to fastq format using the following code (e.g., for CME25A7619408.cram): samtools view -b -@ 33 CME25A7619408.cram > CME25A7619408.bam samtools fastq -@ 4 -1 CME25A7619408_1.r.fastq -2 CME25A7619408_2.r.fastq CME25A7619408.bam cat CME25A7619408_1.r.fastq | paste - - - - | sort -k1,1 -t \" \" | tr \"\t\" \"\n\" > CME25A7619408_1.fastq cat CME25A7619408_2.r.fastq | paste - - - - | sort -k1,1 -t \" \" | tr \"\t\" \"\n\" > CME25A7619408_2.fastq For each sample, RNA separation was performed using biotinylated oligo-dT30VN-tailed oligonucleotides (IDT) conjugated to Dynabeads Streptavidin C1 (65001, Invitrogen) in an RNAse-inhibitor (RNAsin; N2615, Promega) supplemented buffer solution. Sequencing libraries were prepared according to an adapted version of the NMT protocol previously described. Samples were transferred to a 96-well plate and prepared for both transcriptome and methylome/chromatin accessibility sequencing, by physical separation of genomic DNA (gDNA) from messenger RNA (mRNA).