GSM8645899: humanDLPFC_Control_16; Homo sapiens; RNA-Seq
The DLPFC tissue blocks were cryo-sectioned, and total RNA was extracted from 200 mg of cortical tissue using the RNeasy Mini Kit (QIAGEN). For a subset of replicate samples, dry homogenization was performed using a liquid nitrogen cooled mortar and pestle before RNA extraction. Iso-Seq Express Template Preparation for Sequel and Sequel II Systems, PN 101-763-800 Version 02 (October 2019). We used the NEBNext Single Cell/Low Input cDNA Synthesis & Amplification Module, the Iso-Seq Express Oligo Kit, ProNex beads, and the SMRTbell Express Template Prep Kit 2.0 for library preparation. We used 300 ng of cDNA as our input material and subjected the samples to 12 cycles of amplification; additional cycles were added to samples with low yield. We purified the cDNA using a standard workflow. Most of our samples contained transcripts with a central length of approximately 2 kb. We conducted quality control of the SMRT bell libraries using the Qubit dsDNA HS kit and the Agilent Bioanalyzer High Sensitivity kit. We used the Sequel II primer annealing and polymerase binding kit version 2.0 or 2.1. The samples were sequenced on either the Sequel II or Sequel IIe instrument using the Sequel II sequencing plate 2.0 and the Sequel II SMRT Cell 8M. The sequencing was performed with a movie time of 24 hours and a pre-extension time of 2 hours.