GSM8243177: Skin, CTRL, m4, d0.5; Mus musculus; RNA-Seq
Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5' and containing sequences from 5' to 3' for the Illumina read 1 primer, a 6-bp sample barcode (up to 384), a 10-bp unique molecular identifier (UMI), and an anchored oligo(dT)30 for priming; and (3) 9 µl of RT mix containing 4 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 2 pmoles of template switching oligo (TSO), and 0.25 µl of Maxima H Minus Reverse Trascriptase (Thermo Scientific, EP0753). First, barcoded RT primers were added to RNA, which were then denatured at 72°C for 1 min and snap cooled on ice. Second, the RT mix was added, and plates were incubated at 42°C for 120 min. For each library, double stranded cDNA from up to 384 samples were pooled using DNA Clean & Concentrator-5 columns (Zymo Research, D4013), and residual RT primers were removed using exonuclease I (New England Biolabs, M0293). Full-length cDNAs were amplified with 5 to 8 cycles of single-primer PCR using the Advantage 2 PCR Kit (clontech 639206) and cleaned up using SPRIselect magnetic beads (Beckman Coulter B23318). cDNA was quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific 32851) and 50 ng of cDNA per pool of samples was tagmented using the Tagment DNA Enzyme I (Illumina 20034197) and amplified using the NEBNext Ultra II Q5 Master Mix (NEW ENGLAND BioLabs M0544L). Libraries were gel purified using 2% E-Gel EX Agarose Gels (ThermoFisher Scientific G402002), quantified with a Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and a Tapestation 4200 (Agilent Technologies), and sequenced on the NextSeq 550 platform (Illumina).