GSM8112243: ARM-seq Day 35 AlkB Positive c305 Organoid 3; Homo sapiens; ncRNA-Seq
Isolation of total RNA from cerebral cortical organoids and stem cells was performed using Direct-Zol RNA MiniPrep Kit (Zymo Research) with TRI Reagent (Molecular Research Center, Inc.). Since a single organoid would yield far less total RNA, approximately 3-8 organoids would be pooled in trizol depending on organoid size for each sample replicate. Manufacturer's recommended volume of TRI Reagent was added to each sample (~1 mL). For RNA purification of stem cells Trizol was directly added to cell culture plates on ice, scraped, and homogenized via pipetting. Organoids in Trizol were broken down via pipetting inside a 1mL eppendorf tube on ice via a syringe. All total RNA was processed using a MirVana miRNA Isolation Kit (Life Technologies), according to the manufacturer's instructions, to select for RNA <200 nt. This was followed by a RNA Clean and Concentrate-25 (Zymo Research). Samples were divided into plus-AlkB experimental treatment and minus-AlkB control as previously described in ARM-seq methods (Cozen, et al., 2015). This was followed by a phenol chloroform cleanup treatment. RNA samples were then used for library preparations. For ARM-seq, libraries were constructed as previously described (Hrabeta-Robinson, et al. Methods Mol Biol. 2017) which utilizes the NEBNext Multiplex Small RNA Library Prep Set (NEB). PCR-amplified libraries were purified using DNA Clean and Concentrate-5 (Zymo Research) and then size-selected (140-250 nts) on a 6% non-denaturing TBE-acrylamide gel. Libraries were eluted from gel pieces using Gel Elution Buffer (NEB) and precipitated using .3 M NaOAc, 80% Ethanol, and 1 uL of Linear Acrylamide (supplied in NEBNext Kit) at final concentration.  Samples were left in -80C freezer overnight to precipitate. Precipitated libraries were then pelleted, washed twice in 80% Ethanol, and resuspended in Pure H2O. Libraries were then quantified using NanoDrop and Agilent DNA High Sensitivity kit. For DM-tRNA-seq, libraries were constructed as using the TGIRT™ Improved Modular Template-Switching RNA-seq Kit (InGex, LLC), which utilizes Illumina-compatible adapters for amplification and sequencing purposes. Libraries were amplified using PCR primers supplied with the NEBNext Small RNA library Prep Set (NEB) and  purified using Agencourt AMPure XP beads (Beckman). Resulting purified sequencing libraries were quantified using NanoDrop and Agilent DNA High Sensitivity kit. Libraries were then pooled equimolarly, and concentrated using a DNA Clean and Concentrate-5 (Zymo Research).  Pooled PCR-amplified libraries were size-selected (140-250 nts) on a 6% non-denaturing TBE-acrylamide gel to remove unwanted primer dimer products. Libraries were eluted from gel pieces using Gel Elution Buffer (NEB) and precipitated using .3 M NaOAc, 80% Ethanol, and 1 uL of Linear Acrylamide (supplied in NEBNext Kit) at final concentration.  Samples were left in -80C freezer overnight to precipitate. Precipitated libraries wer then pelleted, washed twice in 80% Ethanol, and resuspended in Pure H2O.