Sera mag speedbeads

Volume of supernatant or Golgi-enriched fractions equal to 10 µg was used for label-free analysis. Samples were dried down under vacuum centrifugation and resuspended with 1% SDS with TEAB. A modified version of the Single-Pot Solid-Phase-enhanced Sample Preparation (SP3) protocol was used for sample processing [29 (link)]. Briefly, samples were reduced with Tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 5 mM) and then alkylated with iodoacetamide (12.5 mM) before being quenched with DTT (15 mM). Samples were precipitated onto hydrophilic and hydrophobic Sera-Mag SpeedBeads (carboxylate-modified magnetic beads, GE Healthcare) using sixfold volume of ethanol. Samples were washed twice with 80% ethanol and then digested with trypsin (1:25, enzyme:protein) overnight at 37 °C in the presence of 100 mM TEAB. Peptides were removed from the beads with the aid of a magnet, acidified and then dried down and stored at −20 °C prior to analysis.

Purified tagged GshB was cleaned up using SP3 based purification according to previous protocols^{39 (link)}. Samples were first denatured and reduced using 1% SDS, 10 mM DTT, 100 mM HEPES by boiling at 95 °C, 1,000 rpm for 10 minutes. Samples were then cooled and alkylated with 40 mM 2-chloroacetamide (CAA) for 1 hour at RT in the dark. The alkylation reactions were then quenched with 40 mM DTT for 10 minutes and then samples precipitated on to SeraMag Speed Beads (GE Healthcare, USA) with ethanol (final concentration 50% v/v). Samples were shaken for 10 minutes to allow complete precipitation onto beads and then washed three times with 80% ethanol. The beads were resuspended in 100 mM ammonium bicarbonate containing 1 μg lys-C 1/50 (w/w) and digested overnight at 37 °C. Samples were centrifuged at 14,000 g for 5 minutes to pellet the beads and the supernatant collected and desalted using C18 stage tips before being dried for LC-MS analysis.

We tested commercially available polyT beads (Thermo Fisher Scientific dynabeads, catalog no. 61002), or conjugated carboxylate-coated beads (GE Healthcare Sera-Mag SpeedBeads, catalog no. 65152105050250), and followed the manufacturer conjugation protocol with a 25-dT oligo.

Proteins were extracted from purified leukemic and preleukemia cells in 50 mM HEPES buffer, pH8.5 containing 1% SDS (Fisher BioReagents, Pittsburgh, USA) and protease inhibitor (Pierce, Waltham, MA). The lysate was incubated at 37 °C for 30 min followed by reduction and alkylation using 50 mM TCEP and CAA (Sigma), respectively. Protein concentration was estimated using BCA assay (Sigma). Prepared samples were processed for clean-up and trypsin digestion using SP3 through the addition of Sera-Mag Speed Beads (GE Life Sciences, Chicago, IL) (See Supplemental Material for details). The eluted peptides were separated using a 60 min active gradient from 0–34% Buffer B (0.1% FA in 95% acetonitrile) on a EasyNLC 1200 with a 50µPAC column (ThermoScientific) coupled to a Q Exactive HF orbitrap mass spectrometer. Data was acquired by Data-independent acquisition (See Supplemental Material for details). Statistical significance was determined using the Student’s t-test as implemented in Spectronaut. STRING (https://www.string-db.org) was used to determine proteins interacting with KIFC1. Proteins selected from STRING with evidence of direct experimentally-shown interaction were visualized in volcano plot using R with the Log2-transformed intensities and Log10 of the q-value of each protein on the x-axis and y-axis, respectively.

The libraries were prepared by following the protocol developed by Qi et al. (2018). Genomic DNA was extracted from whole blood using GenElute blood genomic DNA kit (NA2010-1KT; Sigma-Aldrich, Shanghai, China). After having assessed DNA sample quantity and quality by NanoDrop 2000, the qualifying DNA samples were used for library construction using the SuperGBS technology [13 (link)]. The restriction enzyme PstI-HF/MspI (R3140/RO106; New England BioLabs, Beijing, China) was utilized to digest genomic DNA (200 ng), and each end of the digestion products was ligated with barcoded adapter using T4 DNA ligase (M0202; New England BioLabs, Beijing, China). Fragments of 300–700 bp were purified and recovered by a modified magnetic bead recovery system of Sera-Mag SpeedBeads (65152105050250; GE Healthcare Life Sciences). After that, fragments containing adapters on both ends were amplified using PCR. Purified sample libraries were quantified using Qubit dsDNA HS Assay Kit (Q3258; Invitrogen, Carlsbad, CA, USA), and concentrations lower than 5 ng/μL were eliminated. Thirty nanograms of each GBS library were pooled. Pooled GBS libraries (30 samples per species; 900 ng per library) were subjected to pair-end sequencing (150 bp) run on NovaSeq 6000 platform (Illumina, San Diego, CA, USA).