GSM7716869: Fish_embryo_mRNA_sphere_PAL_seq_v4; Danio rerio; OTHER
Frog oocytes and embryos were lysed in 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 [1 tablet per 10 ml buffer], and 200 units/ml SUPERase•In) in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri-reagent for RNA isolation. Fish embryos were de-chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. After removing all E3 medium, Tri-Reagent was added to the embryos for RNA isolation. Mouse GV-oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus-oocyte-complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG-induced mice (16 hr), denuded with 3 mg/ml hyaluronidase for 2 min, washed, and then collected in Tri-reagent for RNA isolation. For gene-specific tail-seq of reporter libraries, total RNA with reporter mRNA libraries was ligated to a pre-adenylated 3ʹ adapter directly in most cases, but for the N60 library injected into fish embryos and frog oocytes, the N37-PAS-N17 library injected into fish embryos and frog oocytes, and the CPEmos-N60 and N60(LC)-PASmos libraries injected into frog oocytes, reporter library mRNAs were enriched by anti-sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was 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 or the RNA isolated from oocyte or embryo lysates was ligated to a pre-adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330, 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 KXS037 in a total volume of 12 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 First-Strand Buffer, 500 µM dNTPs, 5 mM dithiothreitol, 1 unit/µl SUPERase•In, and 200 units SuperScript III (Thermo Fisher, 18080044) at 50°C for 1 hr. After reverse transcription, RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min, followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio-Spin P-30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer (Supplemental using the KAPA HiFi HotStart Kits following the manufacturer's suggested protocol for 10–15 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. For sequencing of endogenous mRNA poly(A)-tail length, libraries were prepared with PAL-seq v3 (for frog oocytes) or PAL-seq v4 (for frog embryos, fish embryos, and mouse oocytes) as described previously (PMID: 34213414). When preparing the sequencing libraries of mRNAs from fish embryos, a different 3ʹ adapter (KXS013) was used for 3ʹ-end ligation, and poly(A)-selected mRNA from HeLa cells was used as spike-in, replacing poly(A)-selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads 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. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined, precipitated with ethanol, and resuspended in 30 µl water. The oligo-depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample after monitoring consistency.