GSM5505416: K-562 STORM-seq cell 66 [H4]; Homo sapiens; RNA-Seq
BULK TOTAL RNA: Total RNA from 5 million K562 cells were isolated using TRIzol (Invitrogen). STORM-seq: total RNA extraction is integrated into the library preparation protocol with Takara Bio's lysis buffer that comes in the SMART-seq Stranded kit (Takara Bio, USA) BULK TOTAL RNA: Libraries were prepared by the Van Andel Genomics Core from 500 ng of total RNA using the KAPA RNA HyperPrep Kit with RiboseErase (v1.16) (Kapa Biosystems, Wilmington, MA USA). RNA was sheared to 300-400 bp. Prior to PCR amplification, cDNA fragments were ligated to Illumina UDIs. Quality and quantity of the finished libraries were assessed using a combination of Agilent DNA High Sensitivity chip (Agilent Technologies, Inc.) and QuantiFluor® dsDNA System (Promega Corp., Madison, WI, USA). SMART-seq STRANDED: Single-cell K562 libraries were prepared using the SSS Kit, following the manufacturer instructions with modifications. Following sorting into PBS, Takara Shearing Master Mix was added to all samples to a final concentration recommended by the kit. The samples underwent 6 minute fragmentation time at 85 °C. Section B step 4 in the protocol was modified to account for the incorporation of UDIs (Takara SMARTer RNA Unique Dual Index Kit-96A, Takara Bio, USA). A total of 2 uL of each UDI was used instead of the recommended 1 uL each of the 5' and 3' PCR primers. For PCR1, 10 cycles of amplification were performed, followed by pooling according to Appendix A. The remainder of the protocol was performed according to manufacturer recommendations. STORM-seq: Single cells were prepared using the SMART-Seq Stranded Kit (Takara USA) on the SPT Labtech Mosquito (HV) at 1:6, then 1:4 dilutions relative to the recommended volumes for subsequent steps in the protocol. In contrast to the manually prepared K562 libraries, for the miniaturized protocol, cells were sorted into a 384-well plate (Eppendorf twin-tec) containing a combination of Shearing Master Mix plus PBS and immediately transferred to a thermocycler and fragmented for 3 minutes at 85 °C. Following 1st Strand Synthesis, PCR1 (10 cycles of amplification) was performed with the addition of SMARTer RNA Unique Dual Index Set A (Takara SMARTer RNA Unique Dual Index Kit-96A) instead of Takara 5' and 3' PCR Primers. Samples were pooled across rows into groups of 12 cells according to Appendix A: Pooling Strategy for Single-Cell Applications and depletion of ribosomal cDNA was performed as indicated. 13 cycles of amplification were performed for PCR2 and the final library was eluted in 13ul 10mM Tris-HCl pH 8.0.