title: GSM8597653: F044_single_nucleotide_mutagenesis_library_frog_oocyte_progesterone_7h_Tail_seq; Xenopus laevis; OTHER
library_construction_protocol: Frog oocytes (100 as a group) were collected at the indicated time after progesterone treatment. OR-2 buffer was removed and oocytes were washed three times with ice-cold buffer RL (20 mM HEPES pH 7.5, 100 mM KCl, 5 mM MgCl2, 1% (v/v) Triton X-100, 100 µg/ml cycloheximide, cOmplete protease inhibitor cocktail [MilliporeSigma, 11836170001, 1 tablet per 10 ml buffer], 10 µl/oocyte). After removing all wash buffer, oocytes were lysed in buffer RL (10 µl per oocyte) supplemented with 200 units/ml SUPERase•In (10 µl/oocyte) by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was mixed with 3x volume TRIzol LS (Thermo Fisher, 10296010). Note that the “0 h post progesterone treatment” sample was prepared by collecting the supernatant of the lysate of untreated oocytes and combining it with the uninjected mRNA library before the Tri-Reagent was added to the mix. All RNAs isolated with the TRIzol-LS were performed with Phasemaker tubes (Thermo Fisher, A33248) according to the manufacturer's protocol. Total RNAs were resuspended in 40 µl water (0.4 µl/oocyte). Total RNAs (10 µl, ~30 µg) isolated from injected oocytes were combined with 10 amol with reporter mRNA poly(A) tail-length standard mix and then mixed with 8 pmol KXSH009, 8 pmol KXSH010, and 2x SSC (0.3 M NaCl, 30 mM sodium citrate pH 7.0) in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads (Thermo Fisher, 65002) and incubated for 20 min at 23°C on a thermal mixer, shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl) and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined, precipitated with ethanol, and resuspended in 6.5 µl water. The anti-sense oligo-enriched RNA was ligated to a pre-adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS347a, 50 mM HEPES pH 7.5, 10 mM MgCl2, 10 mM dithiothreitol, 1 unit/µl T4 RNA ligase 1 (New England Biolabs, M0204S). The ligation reaction was incubated at 23°C for 150 min. After ligation, RNA was extracted with phenol/chloroform, precipitated with ethanol, and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS348 and 1 µl 10 mM dNTPs in a total volume of 13 µl, incubated at 65°C for 5 min, and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x SSIV Buffer, 5 mM dithiothreitol, 1 unit/µl SUPERase•In, and 200 units SuperScript IV Reverse Transcriptase (ThermoFisher, 18090050) at 55°C for 15 min. After reverse transcription, RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min followed by neutralization with 24 µl 1 M HEPES pH 7.5. The cDNA library was ethanol-precipitated, resuspended in water, and amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer (KXS057) that hybridized to a constant region of the library using the KAPA HiFi HotStart Kits following the manufacturer's suggested protocol for 11~14 cycles. The PCR-amplified library was cleaned up twice with AMPure XP beads (Beckman Coulter, A63881) with a beads-to-sample ratio of 1.2. Sequencing was performed on an Illumina HiSeq 2500, with a custom run of two reads. The first read was sequenced with the primer KXS067 for 281 cycles. The second read was sequenced in the same direction with a standard TruSeq sequencing primer for 10 cycles. Additionally, to build a reference sequence, the N60(LC)-PASmos library made from the 0 h post progesterone sample (uninjected) was also sequenced on AVITI, with a custom run of two reads in the same direction for 299 cycles and 10 cycles respectively, using the same primers as those for the HiSeq.