GSM7734538: RNA, eG1-4, rep2; Mus musculus; RNA-Seq
50'000 nuclei were isolated following the low cell input nuclei isolation protocol of the 10x protocol for cell lines and PBMCs. Briefly, cells were resuspended in 500 µl lysis buffer composed of 10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1% BSA, 0.1% Tween, 1 mM DTT, 0.5U/µl Rnase inhibitor, 0.1% NP-40, and 0.01% Digitonin. After incubation for 5 min on ice, 500 µl wash buffer was added (same as lysis buffer but without NP-40 and digitonin) and nuclei were spun down. The washing step was repeated for a total of three times. RNA extraction was done using the Rneasy Plus Micro Kit (Qiagen, 74034) with the following adjustments. Nuclei were resuspended in 350 µl lysis buffer comprising 40 mM DTT. Upon gDNA elimination, the samples were washed with ethanol and transferred to the RNeasy MinElute column. Next, 350 µl of RW1 buffer was added, and samples were spun down. The flow-through was discarded and a mix of 70 µl buffer RDD with 10 µl DNAse was added to the columns. 15 minutes later, 350 µl buffer RW1 was added to the columns and the manufacturer's protocol was followed. Sequencing libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB, E7760S), and sequenced on a NextSeq 500 using 75-nucleotide read length paired-end sequencing. Libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB, E7760S)