GSM6908979: batch4_C13c; Homo sapiens; RNA-Seq
Single cells were mouth-pipetted into PCR tubes that contained 6.75 µl nucleus isolation buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, IGEPAL CA-630 0.3%, 0.1% Bovine serum albumin, 2 mM dithiothreitol) and 0.25 µl Dynabeads™ MyOne™ Silane beads. After incubation on ice for 30 min, sample tubes were gently vortexed for 1 min. Sample tubes were then centrifuged at 500 r.c.f. for 5 min at 4 °C. Afterwards, 5.4 µl supernatant containing cytoplasmic RNA was transferred into a new tube for scRNA sequencing using Smart-seq2 library preparation (see further below), leaving the bead pellet containing the nuclei undisturbed.   After the nucleus isolation, 0.4 µl nick repair mix (0.25 ng/ml linear spike-in DNA, 0.25 ng/ml circular spike-in DNA, 5X NEBNext FFPE DNA Repair Buffer, 0.1225X NEBNext FFPE DNA Repair Mix) was added into each tube containing the bead pellets. Sample tubes were incubated at 20 °C for 1 h after gentle vortexing.  After nick repair, 1.54 µl nuclear lysis mix (40 mM Tris-HCl, 40 mM NaCl, 0.2% TritonX-100, 30 mM dithiothreitol, 2 mM EDTA, 1.6 µg/µl QIAGEN Protease) was added into each sample tube containing repaired product. After gently vortexing, the sample tubes were incubated at 50 °C for 30 min and held at 4 °C. Next, 0.5 µl diluted inhibition mix (10X Protease Inhibitor cocktail (Sigma-Aldrich)) was added to each tube, and tubes were incubated at 37 °C for 1 h.  The lysed products were then subjected to linear DNA digestion. 1.2 µl digestion mix (8.3 mM ATP, 4.16X Plasmid-Safe Reaction Buffer (Lucigen), 0.83-2.49 U/µl Plasmid-Safe ATP-Dependent DNase (Lucigen), 2.08 mM dithiothreitol) was added into each tube, and samples were incubated at 37 °C for 20 h, 70 °C for 10 min and held at 4 °C. Next, 5 µl amplification mix (2X Phi29 buffer (NEB), 2 mM dNTP, 100 µM Exo-Resistant Random Primer (Invitrogen), 0.002 U/µl Pyrophosphatase inorganic (Invitrogen), 1.6 U/µl Phi29 DNA Polymerase (NEB)) was added into each tube, and the sample tubes were incubated at 30 °C for 2-4 h, 65 °C for 10 min and held at 4 °C.   The amplified DNA was purified with DNA Clean & Concentrator-5 (Zymo), after which 10 ng of the purified DNA was subjected to the Nextera XT DNA Library Preparation Kit (Illumina) following the manufacturer's instructions.  